Compounds that induce degradation of anti-apoptotic bcl-2 family proteins and the uses thereof

ABSTRACT

The present disclosure provides compositions and methods for selectively killing senescent cells, wherein the composition comprises a compound of Formula (I) or a compound of Formula (II). The selective killing of senescent cells may delay aging and/or treat age-related disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/325,856, filed Apr. 21, 2016, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions that induce thedegradation of anti-apoptotic Bcl-2 family proteins and their method ofuse in the treatment of various cancers and treatment and prevention ofdiseases and pathologies related to accumulation of senescent cellsduring aging, such as aging, cancer, chronic obstructive pulmonarydisease (COPD), osteoarthritis, atherosclerosis, neurodegenerativediseases, diabetes, and many others. The present invention also relatesto pharmaceutical compositions containing these compounds as well asvarious uses thereof.

BACKGROUND OF THE INVENTION

Aging is the major risk factor for most functional deficits and manydiseases in human, such as cancers, osteoarthritis, osteoporosis,atherosclerosis, neurodegenerative diseases, and diabetes. An increasingbody of evidence demonstrates that aging is associated with anaccumulation of senescent cells (Campisi, Cell 120:513-522, 2005;Campisi, Curr. Opin. Genet. Dev. 21:107-112, 2011; Rodier and Campisi,J. Cell Biol. 192:547-556, 2011). Senescent cell accumulation in tissuesand organs is believed to cause tissue degradation and loss of functiondue to the increased levels of free radicals and various inflammatorymediators produced by senescent cells. Therefore, selective depletion ofsenescent cells may be a novel anti-aging strategy that may preventcancer and various human diseases associated with aging and rejuvenatethe body to live a healthier lifespan. This hypothesis is supported byrecent findings that selective elimination of p16^(Ink4a) (p16)-positivesenescent cells in BubR1 hypomorphic progeroid mouse model via a geneticapproach extended the animals' healthy lifespan by delaying the onset ofseveral age-related pathologies, such as cataracts, sarcopenia, andlordokyphosis (Baker et al., Nature 479:232-236, 2011; Baker et al.,Nature 530.184-189, 2016). These studies validated the great therapeuticpotential of targeting senescent cells.

The Bcl-2 (B-cell lymphoma-2) family of proteins is a group of regulatorproteins that plays a central role in regulating cell death by eitherinducing (pro-apoptotic) or inhibiting (anti-apoptotic) apoptosis.Anti-apoptotic Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bcl-W,and Mcl-1, has been proven to be an attractive target for thedevelopment of novel anti-cancer agents (Lessene et al., Nat. Rev. DrugDiscov. 7:989-1000, 2008; Vogler et al., Cell Death Differ. 2009;16:360-367; Delbridge et al., Nat. Rev. Cancer 16:99-109, 2016).Numerous Bcl-2 small molecule inhibitors have been reported (Bajwa etal., Expert Opin. Ther. Patents 22:37-55, 2012; Vogler, Adv. Med. 1-14,2014). The following are some of the Bcl-2 small molecule inhibitorsthat have been investigated at various stages of drug development:ABT-737 (US20070072860), navitoclax (ABT-263, WO2009155386), venetoclax(ABT-199, WO2010138588), obatoclax (GX 15-070, WO2004106328),(−)-gossypol (AT-101, WO2002097053), sabutoclax (BI-97C1, WO2010120943),TW-37 (WO2006023778), BM-1252 (APG-1252), and A-1155463 (WO2010080503).Venetoclax, a selective Bcl-2 inhibitor, was approved by the FDA inApril 2016 for the treatment of chronic lymphocytic leukemia with 17-pdeletion.

The Bcl-2 family of proteins has also been found to be a potentialtarget for the development of “senolytic” drugs, drugs that targetingsenescent cells for the delay of aging or treatment of aging-associateddisease. For example, navitoclax (ABT-263), an inhibitor of Bcl-2,Bcl-xL, and Bcl-W, has been shown to selectively kill senescent cells inculture and deplete senescent cells in aged mice (WO2015171591; Chang etal., Nat. Med. 22:78-83, 2016; Zhu et al., Aging Cell 2016).

Thus, there is a need in the art to develop compounds capable ofselectively targeting senescent cells and degrading the Bcl-2 family ofproteins.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a compound comprisingFormula (II):

wherein

R¹ is selected from the group consisting of:

R³ is absent, a bond, or a substituted or unsubstituted C₁-C₁₀ alkyl;

A is absent, a bond, a substituted or unsubstituted C₁-C₆ aryl, asubstituted or unsubstituted C₁-C₆ cycloalkyl, or a substituted orunsubstituted C₁-C₆ heterocyclic group;

R⁴ is a bond or a substituted or unsubstituted C₁-C₁₀ alkyl;

n is an integer from 0 to 5;

R² is selected from the group consisting of

The invention also encompasses a method of killing one or more senescentcells in a subject. The method comprises administering a therapeuticallyeffective amount of a compound of the invention to a subject in needthereof.

In another aspect, the invention encompasses a method for delaying atleast one feature of aging in a subject. The method comprisesadministering a therapeutically effective amount of a compound of theinvention to a subject in need thereof.

In yet another aspect, the invention encompasses a method of treating anage-related disease or condition. The method comprises administering atherapeutically effective amount of a compound of the invention to asubject in need thereof.

In still yet another aspect, the invention encompasses a method ofkilling therapy-induced senescent cells. The method comprisesadministering a therapeutically effective amount of a compound of theinvention to a subject in need thereof who has received DNA-damagingtherapy and killing therapy induced-senescent cells in normal and tumortissues following DNA-damaging therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B depicts graphs that show XZ-13906 (2 μM) depletesBcl-xL in normal WI38 (NC-WI38) and ionizing radiation induced senescentWI38 (IR-SC WI38 cells).

FIG. 2A and FIG. 2B depicts graphs show that compound 11 (XZ-13861)(FIG. 2A) and XZ-13906 (FIG. 2B) selectively inhibits IR-SC WI38 cellsbut not normal WI38 cells in a dose-dependent manner.

FIG. 3A and FIG. 3B depicts graphs that show that XZ-14439 dosedependent (FIG. 3A) and time dependently (FIG. 3B) depletes Bcl-xL inIR-SC WI38 cells.

FIG. 4A and FIG. 4B depicts graphs that show that XZ-15416, XZ-15405,XZ-15418, XZ-15421, and PZ-15227 deplete Bcl-xL in IR-SC WI38 (FIG. 4A)and RS4; 11 (FIG. 4B) cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds which are capable ofdegrading the Bcl-2 family of proteins. The bivalent compounds connect aBcl-2 small molecule inhibitor or ligand to an E3 ligase binding moiety,such as cereblon (CRBN) E3 ligase binding moiety (thalidomidederivatives such as pomalidomide) or von Hippel-Landau (VHL) E3 ligasebinding moiety (such as HIF-1α-derived (R)-hydroxyproline containing VHLE3 ligase ligands). CRBN is part of the cullin-4 (CUL4) containing E3ubiquitin ligase complex CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN.Thalidomide and its derivatives, such as lenalidomide and pomalidomide,interact specifically with this CRBN complex and inducing degradation ofessential IKAROS transcription factors. VHL is part of the cullin-2(CUL2) containing E3 ubiquitin ligase complex elongin BC-CUL2-VHL (knownas CRL2VHL) responsible for degradation of the transcription factorHIF-1α. (R)-Hydroxyproline containing VHL E3 ligase ligands derived fromHIF-1α have been identified with high affinity. The bivalent compoundscan actively recruit the Bcl-2 family of proteins to an E3 ubiquitinligase, such as CRBN or VHL E3 ligase, resulting in their degradation byubiquitin proteasome system.

Applicants have discovered that compounds comprising a moiety thatselectively binds to an E3 ubiquitin ligase and a moiety thatselectively binds a target protein, results in ubiquitination andsubsequent degradation of the target protein through the ubiquitinproteasome system. Accordingly, the present disclosure providescompositions and methods for selectively degrading the Bcl-2 family ofproteins. Additional aspects of the invention are described below.

I. Compositions

In an aspect, a composition of the invention comprises a compound ofFormula (I) or a compound of Formula (II). Derivatives of Formula (I) orFormula (II) may be made to improve potency, bioavailability,solubility, stability, handling properties, or a combination thereof, ascompared to an unmodified version.

A composition of the invention may optionally comprise one or moreadditional drugs or therapeutically active agents in addition to acompound of Formula (I) or a compound of Formula (II). A composition ofthe invention may further comprise a pharmaceutically acceptableexcipient, carrier or diluent. Further, a composition of the inventionmay contain preserving agents, solubilizing agents, stabilizing agents,wetting agents, emulsifiers, sweeteners, colorants, odorants, salts(substances of the present invention may themselves be provided in theform of a pharmaceutically acceptable salt), buffers, coating agents orantioxidants.

(a) Compounds of Formula (I)

Provide herein are compounds comprising Formula (I):

R₁-L-R₂  (I)

wherein

-   -   R₁ is a protein targeting unit which binds to one or more        anti-apoptotic Bcl-2 family of proteins;    -   L is a linker unit which covalently links R₁ and R₂ through an        alkyl, branched alkyl, ether, thioether, ester, amine, amide,        carbamate, carbamide, sulfone, aryl, heteroaryl, cycloalkyl, or        heterocyclic group, both end can be same or different; the        linker unit could contain a combination of two or more groups        among alkyl, branched alkyl, ether, thioether, ester, amine,        amide, carbamate, carbamide, sulfone, aryl, heteroaryl,        cycloalkyl, and heterocyclic groups; the linker unit comprises a        length of 1-30 atoms in shortest length; and    -   R₂ is an E3 ubiquitin ligase binding unit which binds to the        CRBN or VHL E3 ubiquitin ligase.

(b) Compounds of Formula (II)

The compounds described by Formula (II) are a subset of the compoundsdescribed by Formula (I). Thus, R₁ and R₂ in Formula (I) are equivalentto R₁ and R₂ in Formula (II), respectively. The L in Formula (I) isdefined as the following in Formula (II):

Also provided herein are compounds comprising Formula (II) or an isomerthereof:

wherein

-   -   R¹ is selected from the group consisting of:

-   -   R³ is absent, a bond, or a substituted or unsubstituted C₁-C₁₀        alkyl;    -   A is absent, a bond, a substituted or unsubstituted C₁-C₆ aryl,        a substituted or unsubstituted C₁-C₆ cycloalkyl, a substituted        or unsubstituted C₁-C₆ heterocyclic group;

R⁴ is a bond or a substituted or unsubstituted C₁-C₁₀ alkyl;

n is an integer from 0 to 5;

R² is selected from the group consisting of

In an embodiment, a compound of Formula (II) comprises any of thepreceding compounds of Formula (II), wherein R¹ may be

In an embodiment, a compound of Formula (II) comprises any of thepreceding compounds of Formula (II), wherein R³ may be absent, anunsubstituted C₁-C₆ alkyl, or a substituted or unsubstituted C₃-C₆ketone.

In a preferred embodiment, a compound of Formula (II) comprises any ofthe preceding compounds of Formula (II), wherein R³ may be absent, abond, an unsubstituted C₁-C₃ alkyl, or an unsubstituted C₃-C₆ ketone.

In still a preferred embodiment, a compound of Formula (II) comprisesany of the preceding compounds of Formula (II), wherein R³ may beabsent, a bond, 2-pentanone, or an unsubstituted C₂-C₃ alkyl.

In another embodiment, a compound of Formula (II) comprises any of thepreceding compounds of Formula (II), wherein A may be absent, a bond, ora substituted or unsubstituted C₁-C₆ heterocyclic group.

In a preferred embodiment, a compound of Formula (II) comprises any ofthe preceding compounds of Formula (II), wherein A may be absent, abond, or an unsubstituted C₅ heterocyclic group.

In still a preferred embodiment, a compound of Formula (II) comprisesany of the preceding compounds of Formula (II), wherein A may be absent,a bond, or a triazole.

In another embodiment, a compound of Formula (II) comprises any of thepreceding compounds of Formula (II), wherein n may be 0 to 3.

In a preferred embodiment, a compound of Formula (II) comprises any ofthe preceding compounds of Formula (II), wherein n may be 0 to 2.

In still a preferred embodiment, a compound of Formula (II) comprisesany of the preceding compounds of Formula (II), wherein n may be 1 to 2.

In another embodiment, a compound of Formula (II) comprises any of thepreceding compounds of Formula (II), wherein R⁴ may be a bond or asubstituted or unsubstituted C₁-C₁₀ alkyl.

In a preferred embodiment, a compound of Formula (II) comprises any ofthe preceding compounds of Formula (II), wherein R⁴ may be a bond or asubstituted C₁-C₁₀ alkyl.

In another embodiment, a compound of Formula (II) comprises any of thepreceding compounds of Formula (II), wherein R² is

In one embodiment, a compound of the disclosure comprises Formula (II),wherein R¹ may be

R³ may be absent, an unsubstituted C₁-C₆ alkyl, or a substituted orunsubstituted C₃-C₆ ketone; A may be absent, a bond, or a substituted orunsubstituted C₁-C₆ heterocyclic group; n may be 0 to 3; R⁴ may be abond or a substituted or unsubstituted C₁-C₁₀ alkyl; and R² may be

In another embodiment, a compound of the disclosure comprises Formula(II), wherein R¹ may be

R³ may be absent, an unsubstituted C₁-C₆ alkyl, or a substituted orunsubstituted C₃-C₆ ketone; A may be absent, a bond, or a substituted orunsubstituted C₁-C₆ heterocyclic group; n may be 0 to 3, R⁴ may be abond or a substituted or unsubstituted C₁-C₁₀ alkyl; and R² may be

In still another embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be absent, a bond, 2-pentanone, or an unsubstituted C₂-C₃ alkyl;B may be absent, a bond, or a substituted or unsubstituted C₁-C₆heterocyclic group; n may be 0 to 3; R⁴ may be a bond or a substitutedor unsubstituted C₁-C₁₀ alkyl; and R² may be

In still another embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be absent, an unsubstituted C₁-C₆ alkyl, or a substituted orunsubstituted C₃-C₆ ketone; A may be absent, a bond, or a triazole; nmay be 0 to 3; R⁴ may be a bond or a substituted or unsubstituted C₁-C₁₀alkyl; and wherein R² may be

In still another embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be absent, an unsubstituted C₁-C₆ alkyl, or a substituted orunsubstituted C₃-C₆ ketone; A may be absent, a bond, or a substituted orunsubstituted C₁-C₆ heterocyclic group; n may be 1 to 2; R⁴ may be abond or a substituted or unsubstituted C₁-C₁₀ alkyl; and wherein R² maybe

In still another embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be absent, an unsubstituted C₁-C₆ alkyl, or a substituted orunsubstituted C₃-C₆ ketone; A may be absent, a bond, or a substituted orunsubstituted C₁-C₆ heterocyclic group; n may be 0 to 3; R⁴ may be abond or a substituted C₁-C₁₀ alkyl, and R² may be

In a different embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be absent, an unsubstituted C₁-C₆ alkyl, or a substituted orunsubstituted C₃-C₆ ketone; A may be absent, a bond, or a substituted orunsubstituted C₁-C₆ heterocyclic group; n may be 0 to 3; R⁴ may be abond or a substituted or unsubstituted C₁-C₁₀ alkyl; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 2, A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 1; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be propyl; n may be 2; A may be a triazole; R⁴ may be a bond; andR² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 3; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 1; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C₃-alkyl; n may be 3; A may be a triazole; R⁴ may be a bond;and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 2; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 1; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be propyl; n may be 2; A may be a triazole; R⁴ may be a bond; andR² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be butan-1-amine; A may be absent; n may be 2; R⁴ may beN-(4-ethylamino)butyl)acetamide; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 2; A may be a triazole; R⁴ may be C(O);and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 2; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 2; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; n may be 1; A may be a triazole; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O)NH; n may be 1; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(S)NH; n may be 1; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O); n may be 1; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O); n may be 2; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O); n may be 3; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O); n may be 0; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; n may be 1; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O)CH₂; n may be 1; A may be a triazole; R⁴ may be a bond;and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O)NH; n may be 1; A may be a bond; R⁴ may be (CH₂)₂C(O)NH;and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O); n may be 2; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O)NH; n may be 1; A may be a bond; R⁴ may be (CH₂)₂C(O)NH;and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; n may be 0; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; n may be 1; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be C(O)NH; n may be 1; A may be absent; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R¹ may be a bond; A may be absent; n may be 2; R⁴ may beN-(4-ethylamino)butyl)acetamide; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; A may be absent; n may be 2; R⁴ may beN-(4-ethylamino)butyl)acetamide; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; A may be absent; n may be 2; R⁴ may beN-(4-ethylamino)butyl)acetamide; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; A may be absent; n may be 2; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; A may be a triazole; n may be 2; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be 2-pentanone; A may be a triazole; n may be 2; R⁴ may be abond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be N-ethylpropionamide; A may be a triazole; n may be 2; R⁴ maybe a bond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be propyl; A may be triazole; n may be 3; R⁴ may be a bond; andR² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; A is absent; n may be 3; R⁴ may beN-(4-(ethylamino)butyl)acetamide; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be propyl; A is a triazole; n may be 2; R⁴ may be a bond; and R²may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a bond; A is absent; n may be 2; R⁴ may beN-(4-(ethylamino)butyl)acetamide; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may be a propyl; A is a triazole; n may be 2; R⁴ may be a bond; andR² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may by 2-pentanone; A may be triazole; n may be 2; R⁴ may be a bond;and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may by N-methylacetamide; A may be a triazole; n may be 2; R⁴ may bea bond; and R² may be

In a preferred embodiment, a compound of the disclosure comprisesFormula (II), wherein R¹ may be

R³ may by 2-pentanone; A may be a triazole; n may be 1; R⁴ may be abond; and R² may be

In an exemplary embodiment, a compound of Formula (II) comprises any ofthe preceding compounds of Formula (II), may be selected from the groupconsisting of:

(c) Components of the Composition

The present disclosure also provides pharmaceutical compositions. Thepharmaceutical compositions comprise a compound of Formula (I) or acompound of Formula (II), as an active ingredient and at least onepharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylam ides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate,citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally, parenterally, or topically indosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, or intrasternal injection, or infusion techniques.Formulation of drugs is discussed in, for example, Gennaro, A. R.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(18th ed, 1995), and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).In a specific embodiment, a composition may be a food supplement or acomposition may be a cosmetic.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, and intraperitoneal), the preparation may bean aqueous or an oil-based solution. Aqueous solutions may include asterile diluent such as water, saline solution, a pharmaceuticallyacceptable polyol such as glycerol, propylene glycol, or other syntheticsolvents; an antibacterial and/or antifungal agent such as benzylalcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and thelike; an antioxidant such as ascorbic acid or sodium bisulfite; achelating agent such as etheylenediaminetetraacetic acid; a buffer suchas acetate, citrate, or phosphate; and/or an agent for the adjustment oftonicity such as sodium chloride, dextrose, or a polyalcohol such asmannitol or sorbitol. The pH of the aqueous solution may be adjustedwith acids or bases such as hydrochloric acid or sodium hydroxide.Oil-based solutions or suspensions may further comprise sesame, peanut,olive oil, or mineral oil.

The compositions may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carried, for example water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Pharmaceutical compositions adapted fortopical administration may be formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In some embodiments, the pharmaceutical composition is appliedas a topical ointment or cream. When formulated in an ointment, theactive ingredient may be employed with either a paraffinic or awater-miscible ointment base. Alternatively, the active ingredient maybe formulated in a cream with an oil-in-water cream base or awater-in-oil base. Pharmaceutical compositions adapted for topicaladministration to the eye include eye drops wherein the activeingredient is dissolved or suspended in a suitable carrier, especiallyan aqueous solvent. Pharmaceutical compositions adapted for topicaladministration in the mouth include lozenges, pastilles and mouthwashes. Transmucosal administration may be accomplished through the useof nasal sprays, aerosol sprays, tablets, or suppositories, andtransdermal administration may be via ointments, salves, gels, patches,or creams as generally known in the art.

In certain embodiments, a composition a compound of Formula (I) or acompound of Formula (II) is encapsulated in a suitable vehicle to eitheraid in the delivery of the compound to target cells, to increase thestability of the composition, or to minimize potential toxicity of thecomposition. As will be appreciated by a skilled artisan, a variety ofvehicles are suitable for delivering a composition of the presentinvention. Non-limiting examples of suitable structured fluid deliverysystems may include nanoparticles, liposomes, microemulsions, micelles,dendrimers and other phospholipid-containing systems. Methods ofincorporating compositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery a compound of Formula (I) or a compound of Formula (II) in viewof their structural and chemical properties. Generally speaking,liposomes are spherical vesicles with a phospholipid bilayer membrane.The lipid bilayer of a liposome may fuse with other bilayers (e.g., thecell membrane), thus delivering the contents of the liposome to cells.In this manner, a compound comprising Formula (I) or a compoundcomprising Formula (II) may be selectively delivered to a cell byencapsulation in a liposome that fuses with the targeted cell'smembrane.

Liposomes may be comprised of a variety of different types ofphosolipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate),n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palm itoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide,1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying a compound of Formula (I) or a compound of Formula(II) (i.e., having at least one methionine compound) may be prepared byany known method of preparing liposomes for drug delivery, such as, forexample, detailed in U.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561,4,755,388, 4,828,837, 4,925,661, 4,954,345, 4,957,735, 5,043,164,5,064,655, 5,077,211 and 5,264,618, the disclosures of which are herebyincorporated by reference in their entirety. For example, liposomes maybe prepared by sonicating lipids in an aqueous solution, solventinjection, lipid hydration, reverse evaporation, or freeze drying byrepeated freezing and thawing. In a preferred embodiment the liposomesare formed by sonication. The liposomes may be multilamellar, which havemany layers like an onion, or unilamellar. The liposomes may be large orsmall. Continued high-shear sonication tends to form smaller unilamellarlipsomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of methioninecompound, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. The compoundcomprising Formula (I) or Formula (II) may be encapsulated in amicroemulsion by any method generally known in the art.

(d) Additional Compounds

In an aspect, the composition further comprises at least one or moreanticancer therapeutics.

A chemotherapeutic agent refers to a chemical compound that is useful inthe treatment of cancer. The compound may be a cytotoxic agent thataffects rapidly dividing cells in general, or it may be a targetedtherapeutic agent that affects the deregulated proteins of cancer cells.The chemotherapeutic agent may be an alkylating agent, ananti-metabolite, an anti-tumor antibiotic, an anti-cytoskeletal agent, atopoisomerase inhibitor, an anti-hormonal agent, a targeted therapeuticagent, a photodynamic therapeutic agent, or a combination thereof.

Non-limiting examples of suitable alkylating agents include altretamine,benzodopa, busulfan, carboplatin, carboquone, carmustine (BCNU),chlorambucil, chlornaphazine, cholophosphamide, chlorozotocin,cisplatin, cyclosphosphamide, dacarbazine (DTIC), estramustine,fotemustine, ifosfamide, improsulfan, lipoplatin, lomustine (CCNU),mafosfamide, mannosulfan, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, meturedopa, mustine (mechlorethamine),mitobronitol, nimustine, novembichin, oxaliplatin, phenesterine,piposulfan, prednimustine, ranimustine, satraplatin, semustine,temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine,triethylenephosphoramide (TEPA), triethylenethiophosphaoramide(thiotepa), trimethylolomelamine, trofosfamide, uracil mustard anduredopa.

Suitable anti-metabolites include, but are not limited to aminopterin,ancitabine, azacitidine, 8-azaguanine, 6-azauridine, capecitabine,carmofur (1-hexylcarbomoyl-5-fluorouracil), cladribine, clofarabine,cytarabine (cytosine arabinoside (Ara-C)), decitabine, denopterin,dideoxyuridine, doxifluridine, enocitabine, floxuridine, fludarabine,5-fluorouracil, gemcetabine, hydroxyurea (hydroxycarbamide), leucovorin(folinic acid), 6-mercaptopurine, methotrexate, nafoxidine, nelarabine,oblimersen, pemetrexed, pteropterin, raltitrexed, tegofur, tiazofurin,thiamiprine, tioguanine (thioguanine), and trimetrexate.

Non-limiting examples of suitable anti-tumor antibiotics includeaclacinomysin, aclarubicin, actinomycins, adriamycin, aurostatin (forexample, monomethyl auristatin E), authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, epoxomicin,esorubicin, idarubicin, marcellomycin, mitomycins, mithramycin,mycophenolic acid, nogalamycin, olivomycins, peplomycin, plicamycin,potfiromycin, puromycin, quelamycin, rodorubicin, sparsomycin,streptonigrin, streptozocin, tubercidin, valrubicin, ubenimex,zinostatin, and zorubicin.

Non-limiting examples of suitable anti-cytoskeletal agents includecabazitaxel, colchicines, demecolcine, docetaxel, epothilones,ixabepilone, macromycin, omacetaxine mepesuccinate, ortataxel,paclitaxel (for example, DHA-paclitaxel), taxane, tesetaxel,vinblastine, vincristine, vindesine, and vinorelbine.

Suitable topoisomerase inhibitors include, but are not limited to,amsacrine, etoposide (VP-16), irinotecan, mitoxantrone, RFS 2000,teniposide, and topotecan.

Non-limiting examples of suitable anti-hormonal agents such asaminoglutethimide, antiestrogen, aromatase inhibiting 4(5)-imidazoles,bicalutamide, finasteride, flutamide, fluvestrant, goserelin,4-hydroxytamoxifen, keoxifene, leuprolide, LY117018, mitotane,nilutamide, onapristone, raloxifene, tamoxifen, toremifene, andtrilostane.

Examples of targeted therapeutic agents include, without limit,monoclonal antibodies such as alemtuzumab, cartumaxomab, edrecolomab,epratuzumab, gemtuzumab, gemtuzumab ozogamicin, glembatumumab vedotin,ibritumomab tiuxetan, reditux, rituximab, tositumomab, and trastuzumab;protein kinase inhibitors such as bevacizumab, cetuximab, crizonib,dasatinib, erlotinib, gefitinib, imatinib, lapatinib, mubritinib,nilotinib, panitumumab, pazopanib, sorafenib, sunitinib, toceranib, andvandetanib;

angiogeneisis inhibitors such as angiostatin, bevacizumab, denileukindiftitox, endostatin, everolimus, genistein, interferon alpha,interleukin-2, interleukin-12, pazopanib, pegaptanib, ranibizumab,rapamycin (sirolimus), temsirolimus, and thalidomide; and growthinhibitory polypeptides such as bortazomib, erythropoietin, interleukins(e.g., IL-1, IL-2, IL-3, IL-6), leukemia inhibitory factor, interferons,romidepsin, thrombopoietin, TNF-α, CD30 ligand, 4-1BB ligand, and Apo-1ligand.

Non-limiting examples of photodynamic therapeutic agents include aminolevulinic acid, methyl am inolevulinate, retinoids (alitretinon, tamibarotene, tretinoin), and temoporfin.

Other antineoplastic agents include anagrelide, arsenic trioxide,asparaginase, bexarotene, bropirimine, celecoxib, chemically linked Fab,efaproxiral, etoglucid, ferruginol, lonidamide, masoprocol, miltefosine,mitoguazone, talapanel, trabectedin, and vorinostat.

Also included are pharmaceutically acceptable salts, acids, orderivatives of any of the above listed agents. The mode ofadministration of the chemotherapeutic agent can and will vary dependingupon the agent and the type of tumor or neoplasm. Suitable modes ofadministration were detailed in Section II(d), below. A skilledpractitioner will be able to determine the appropriate dose of thechemotherapeutic agent.

II. Methods

The present disclosure encompasses a method of selectively killing oneor more senescent cells in a sample, the method comprising contacting acomposition comprising an effective amount of a compound of Formula (I)or a compound of Formula (II) with the sample. In another aspect, thepresent disclosure encompasses a method of selectively killing one ormore senescent cells in a subject in need thereof, the method comprisingadministering to the subject a composition comprising a therapeuticallyeffective amount of a compound of Formula (I) or a compound of Formula(II).

The present disclosure encompasses a method of selectively killing oneor more cancer cells in a sample, the method comprising contacting acomposition comprising an effective amount of a compound of Formula (I)or a compound of Formula (II) with the sample. In another aspect, thepresent disclosure encompasses a method of selectively killing one ormore cancer cells in a subject in need thereof, the method comprisingadministering to the subject a composition comprising a therapeuticallyeffective amount of a compound of Formula (I) or a compound of Formula(II).

By selectively killing one or more senescent cells is meant acomposition of the invention does not appreciably kill non-senescentcells at the same concentration. Accordingly, the median lethal dose orLD50 of the inhibitor in non-senescent cells may be about 5 to about 50times higher than the LD50 of the inhibitor in senescent cells. As usedherein, the LD50 is the concentration of inhibitor required to kill halfthe cells in the cell sample. For example, the LD50 of the inhibitor innon-senescent cells may be greater than about 5, about 6, about 7, about8, about 9 or about 10 times higher than the LD50 of the inhibitor insenescent cells. Alternatively, the LD50 of the inhibitor innon-senescent cells may be greater than about 10, about 15, about 20,about 25, about 30, about 35, about 40, about 45, or about 50 timeshigher than the LD50 of the inhibitor in senescent cells. Additionally,the LD50 of the inhibitor in non-senescent cells may be greater than 50times higher than the LD50 of the inhibitor in senescent cells. In aspecific embodiment, the LD50 of the inhibitor in non-senescent cells isgreater than 10 times higher than the LD500 of the inhibitor insenescent cells. In another specific embodiment, the LD50 of theinhibitor in non-senescent cells is greater than 20 times higher thanthe LD50 of the inhibitor in senescent cells.

The progression from an actively dividing cell to a metabolicallyactive, non-dividing cell is termed “senescence” or “cellularsenescence.” As used herein, the terms “senescence” and “cellularsenescence” may be used interchangeably. The term “senescence” alsorefers to the state into which cells enter after multiple rounds ofdivision and, as a result of cellular pathways, future cell division isprevented from occurring even though the cell remains metabolicallyactive. Senescent cells may differ from their pre-senescent counterpartsin one or more of the following ways: 1) they arrest growth and cannotbe stimulated to reenter the cell cycle by physiological mitogens; 2)they become resistant to apoptotic cell death; and/or 3) they acquirealtered differentiated functions.

In contrast to cancer cells which grow and divide uncontrollably, theability of most differentiated eukaryotic cells to proliferate isfinite. Stated another way, normal cells have an intrinsicallydetermined limit to the number of cell divisions through which they canproceed. This phenomenon has been termed “replicative cellularsenescence” and is an intrinsic anticancer mechanism that limits acell's proliferative ability, thereby preventing neoplastictransformation. Another form of senescence is “premature cellularsenescence.” Premature cellular senescence, like replicative cellularsenescence, is a terminal fate of mitotic cells, characterized bypermanent cell cycle arrest. Unlike replicative cellular senescence,however, premature cellular senescence does not require telomeredeterioration and can be induced by a variety of stressors including,but not limited to, ultraviolet light, reactive oxygen species,chemotherapeutics, environmental toxin, cigarette smoking, ionizingradiation, distortion of chromatin structure, excessive mitogenicsignaling, and oncogenic mutations. Still another form of senescence istherapy-induced senescence (TIS) which refers to the phenomenon of asubset of tumor cells being forced into a senescent state by therapeuticagents. TIS is known to develop because of certain treatments, includingradiotherapy and chemotherapy.

The number of senescent cells in various organs and tissues of a subjectincreases with age. The accumulation of senescent cells may drive thedeterioration that underlies aging and age-related diseases. Forexample, the accumulation of senescent cells in aged tissue maycontribute to age-associated tissue dysfunction, reduced regenerativecapacity, and disease. In this context, senescence is considereddeleterious because it contributes to decrements in tissue renewal andfunction. As a non-limiting example, an aged tissue may lack the abilityto respond to stress when proliferation is required thereby resulting inthe reduced fitness seen with aging. A key component of this model isthat substantial numbers of senescent cells should be present in tissueswith aging, without, or prior to, pathology.

(a) Senescent Cells

A senescent cell may be a cell that ceases to divide but remainsmetabolically active. The non-dividing cells may remain viable for manyweeks, but fail to grow/replicate DNA despite the presence of amplespace, nutrients and growth factors in the medium. Thus, the senescencegrowth arrest is essentially permanent because senescent cells cannot bestimulated to proliferate by known physiological stimuli. Further, asenescent cell of the invention may be resistant to certain apoptoticsignals and may acquire widespread changes in gene expression. Theresistance to apoptosis may explain the increase in senescent cells withage. Manipulation of pro- and anti-apoptotic proteins may cause cellsthat are destined to die by apoptosis to senesce and, conversely, causecells that are destined to senesce to undergo apoptosis.

A senescent cell of the invention may be senescent due to replicativecellular senescence, premature cellular senescence or therapy-inducedsenescence. Senescent cells that are senescent due to replication mayhave undergone greater than 60 population doublings. Alternatively,senescent cells that are senescent due to replication may have undergonegreater than 40, greater than 50, greater than 60, greater than 70 orgreater than 80 population doublings. A senescent cell that isprematurely cellular senescent may be induced by, but not limited to,ultraviolet light, reactive oxygen species, chemotherapeutics,environmental toxin, cigarette smoking, ionizing radiation, distortionof chromatin structure, excessive mitogenic signaling, and oncogenicmutations. In a specific embodiment, premature cellular senescence maybe induced by ionizing radiation (IR). In another specific embodiment,premature cellular senescence may also be induced by ectopictransfection with Ras oncogene. A senescent cell that is therapy-inducedsenescent may have been exposed to DNA-damaging therapy.

A senescent cell of the invention may generally be a eurkaryotic cell.Non-limiting examples of senescent cells may include, but are notlimited to, mammary epithelial cells, keratinocytes, cardiac myocytes,chondrocytes, endothelial cells (large vessels), endothelial cells(microvascular), epithelial cells, fibroblasts, follicle dermal papillacells, hepatocytes, melanocytes, osteoblasts, preadipocytes, primarycells of the immune system, skeletal muscle cells, smooth muscle cells,adipocytes, neurons, glial cells, contractile cells, exocrine secretoryepithelial cells, extracellular matrix cells, hormone secreting cells,keratinizing epithelial cells, islet cells, lens cells, mesenchymal stemcells, pancreatic acinar cells, paneth cells of the small intestine,primary cells of hemopoietic linage, primary cells of the nervoussystem, sense organ and peripheral neuron supporting cells, wetstratified barrier epithelial cells and stem cells. In a specificembodiment, the stem cells are adult stem cells. Adult stem cells arestem cells which maintain and repair the tissue in which they are foundand are generally referred to by their tissue of origin. Non-limitingexamples of adult stem cells include muscle stem cells, hematopoieticstem cells, heart stem cells, neural stem cells, mesenchymal stem cells,intestinal stem cells, skin stem cells, adipose-derived stem cells,endothelial stem cells, and dental pulp stem cells. In a specificembodiment, a senescent cell of the invention is a fibroblast. Inanother specific embodiment, a senescent cell may be a hematopoieticstem cell.

Further, a senescent cell of the invention may be found in renewabletissues, including the vasculature, hematopoietic system, epithelialorgans and the stroma. A senescent cell of the invention may also befound at sites of aging or chronic age-related pathology, such asosteoarthritis and atherosclerosis. Further, a senescent cell of theinvention may be associated with benign dysplastic or preneoplasticlesions and benign prostatic hyperplasia. In an embodiment, a senescentcell of the invention may be found in normal and tumor tissues followingDNA-damaging therapy. In a specific embodiment, a senescent cell may befound at a site of aging or age-related pathology.

An age-related pathology may include any disease or condition which isfully or partially mediated by the induction or maintenance of anon-proliferating or senescent state in a cell or a population of cellsin a subject. Non-limiting examples include age-related tissue or organdecline which may lack visible indication of pathology, or overtpathology such as a degenerative disease or a function-decreasingdisorder. For example, Alzheimer's disease, Parkinson's disease,cataracts, macular degeneration, glaucoma, atherosclerosis, acutecoronary syndrome, myocardial infarction, stroke, hypertension,idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonarydisease (COPD), osteoarthritis, type 2 diabetes, obesity, fatdysfunction, coronary artery disease, cerebrovascular disease,periodontal disease, and cancer treatment-related disability such asatrophy and fibrosis in various tissues, brain and heart injury, andtherapy-related myelodysplastic syndromes. Additionally, an age-relatedpathology may include an accelerated aging disease such as progeroidsyndromes (i.e., Hutchinson-Gilford progeria syndrome, Werner syndrome,Bloom syndrome, Rothmund-Thomson Syndrome, Cockayne syndrome, xerodermapigmentosum, trichothiodystrophy, combined xerodermapigmentosum-Cockayne syndrome, and restrictive dermopathy), ataxiatelangiectasia, Fanconi anemia, Friedreich's ataxia, dyskeratosiscongenital, aplastic anemia, IPF, and others. A method of identifying anage-related disease or condition as described herein may includedetecting the presence of senescent cells.

(b) Detecting Senescent Cells

In an aspect, a method of the invention may comprise detecting senescentcells. Senescent cells may be detected in vivo or in vitro. Suitablemarkers for detecting senescent cells in vitro and in vivo are known inthe art. For example, methods to detect senescent cells may include, butare not limited to, detecting lack of DNA replication by incorporationof a DNA-staining reagent (e.g., 5-bromodeoxyuridine (BrdU),3H-thymidine), immunostaining for proteins such as proliferating cellnuclear antigen (PCNA) and Ki-67, histochemical staining forsenescence-associated β-galactosidase (SA-β-gal), detecting expressionof p16, p19, Pai1, Igfbp2, IL-6, Mmp13, Nrg1, differentiatedembryo-chondrocyte expressed-1 (DEC1), p15 (a CDK1) and decoy deathreceptor-2 (DCR2), detecting cytological markers such assenescence-associated heterochromatin foci (SAHFs) andsenescence-associated DNA-damage foci (SDFs). SAHFs may be detected bythe preferential binding of DNA dyes, such as4′,6-diamidino-2-phenylindole (DAPI), and the presence of certainheterochromatin-associated histone modifications (e.g., H3 Lys9methylation) and proteins (e.g., heterochromatin protein-1 (HP1)).Additionally, senescent cells may be detected as described in U.S. Pat.No. 5,491,069 and US Patent Application No. 2010/0086941. In certainembodiments, senescent cells are detected by histochemical staining forSA-β-gal.

In certain embodiments, one or more senescent cells are detected in asample. A sample may be a cell sample, a tissue sample, or a biopsy froma subject. Generally speaking, a sample may be dependent on theage-related pathology. For instance, a sample may be tissue biopsymaterial. As such, a tissue sample may be from esophagus, stomach,liver, gallbladder, pancreas, adrenal glands, bladder, gallbladder,large intestine, small intestine, kidneys, liver, pancreas, colon,stomach, thymus, spleen, brain, spinal cord, nerves, adipose tissue,heart, lungs, eyes, corneal, skin or islet tissue or organs. In aspecific embodiment, a tissue sample may be from lung, skeletal muscle,and brain. In another specific embodiment, a tissue sample may be fromliver and heart. Alternatively, a sample may be a cell sample. As such,a cell sample may be oocytes and/or spermatozoa, mesenchymal stem cells,adipocytes, central nervous system neurons and glial cells, contractilecells, exocrine secretory epithelial cells, extracellular matrix cells,hormone secreting cells, keratinizing epithelial cells, islet cells,kidney cells, lens cells, pancreatic acinar cells, paneth cells of smallintestine, primary cells of hemopoietic lineage, primary cells of thenervous system, sense organ and peripheral neuron supporting cells orwet stratified barrier epithelial cells. Detection of senescent cellsmay be used to assess the replicative history of tissues, therebyproviding a method for evaluation of the physiological, in contrast tothe chronological age of the tissue.

The number of senescent cells may increase with age. The number ofsenescent cells in a tissue or sample may be from less than 1% togreater than 15%. In an embodiment, the number of senescent cells in atissue or sample may be less than 1%, less than 2%, less than 3%, lessthan 4%, or less than 5%. In another embodiment, the number of senescentcells in a tissue or sample may be about 5%, about 6%, about 7%, about8%, about 9%, or about 10%. In still another embodiment, the number ofsenescent cells in a tissue or sample may be greater than 10%, greaterthan 11%, greater than 12%, greater than 13%, greater than 14%, orgreater than 15%.

(c) Measuring Cell Death

In an aspect, a method of the invention may comprise measuring celldeath of senescent cells. Methods of measuring cell death are known inthe art. For example, cell death may be measured by Giemsa staining,trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) doublestaining for fluorescence microscopy and flow cytometry, propidiumiodide (PI) staining, annexin V assay, TUNEL assay, DNA ladder, LDHactivity, and MTT assay. In a preferred embodiment, cell death is due toinduction of apoptosis. Cell death due to induction of apoptosis may bemeasured by observation of morphological characteristics including cellshrinkage, cytoplasmic condensation, chromatin segregation andcondensation, membrane blebbing, and the formation of membrane-boundapoptotic bodies. Cell death due to induction of apoptosis may bemeasured by observation of biochemical hallmarks includinginternucleosomal DNA cleavage into oligonucleosome-length fragments.Traditional cell-based methods of measuring cell death due to inductionof apoptosis include light and electron microscopy, vital dyes, andnuclear stains. Biochemical methods include DNA laddering, lactatedehydrogenase enzyme release, and MTT/XTT enzyme activity. Additionally,terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling of DNA fragments (TUNEL) and in situ end labeling (ISEL)techniques are used, which when used in conjunction with standard flowcytometric staining methods yield informative data relating cell deathto various cellular parameters, including cell cycle and cell phenotype.See Loo and Rillema, Methods Cell Biol. 1998; 57:251-64, which isincorporated herein by reference, for a review of these methods. In anexemplary embodiment, cell death due to apoptosis may be measured by thereduction of procaspase-3. Caspase-3 has been implicated as an“effector” caspase associated with the initiation of the “death cascade”and is therefore an important marker of the cell's entry point into theapoptotic signaling pathway. Caspase-3 is activated by the upstreamcaspase-8 and caspase-9, and since it serves as a convergence point fordifferent signaling pathways, it is well suited as a read-out in anapoptosis assay.

The results of these methods may be used to determine the percentage ofviable cells. In a preferred embodiment, cell death may be measured as areduction in viable cells. Since a composition of the inventionselectively kills senescent cells, a reduction in viable cells isindicative of a reduction in senescent cells. As described in SectionIII(b), the number of senescent cells in a sample may be from less than1% to greater than 15%. As such, a reduction in viable cells followingadministration of an inhibitor of the invention may be greater than 15%to less than 1%. For example, the reduction in viable cells may be lessthan 1%, less than 2%, less than 3%, less than 4%, or less than 5%.Alternatively, the reduction in viable cells may be about 5%, about 6%,about 7%, about 8%, about 9%, or about 10%. Additionally, the reductionin viable cells may be greater than 10%, greater than 11%, greater than12%, greater than 13%, greater than 14%, or greater than 15%.

(d) Administration

In certain aspects, a therapeutically effective amount of a compositionof the invention may be administered to a subject. Administration isperformed using standard effective techniques, including peripherally(i.e., not by administration into the central nervous system) or locallyto the central nervous system. Peripheral administration includes but isnot limited to oral, inhalation, intravenous, intraperitoneal,intra-articular, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. Localadministration, including directly into the central nervous system (CNS)includes but is not limited to via a lumbar, intraventricular orintraparenchymal catheter or using a surgically implanted controlledrelease formulation. The route of administration may be dictated by thedisease or condition to be treated. For example, if the disease orcondition is COPD or IPF, the composition may be administered viainhalation. Alternatively, if the disease or condition isosteoarthritis, then the composition may be administered viaintra-articular invention. It is within the skill of one in the art, todetermine the route of administration based on the disease or conditionto be treated. In a specific embodiment, a composition of the inventionis administered orally.

Pharmaceutical compositions for effective administration aredeliberately designed to be appropriate for the selected mode ofadministration, and pharmaceutically acceptable excipients such ascompatible dispersing agents, buffers, surfactants, preservatives,solubilizing agents, isotonicity agents, stabilizing agents and the likeare used as appropriate. Remington's Pharmaceutical Sciences, MackPublishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition,incorporated herein by reference in its entirety, provides a compendiumof formulation techniques as are generally known to practitioners.

For therapeutic applications, a therapeutically effective amount of acomposition of the invention is administered to a subject. A“therapeutically effective amount” is an amount of the therapeuticcomposition sufficient to produce a measurable response (e.g., celldeath of senescent cells, an anti-aging response, an improvement insymptoms associated with a degenerative disease, or an improvement insymptoms associated with a function-decreasing disorder). Actual dosagelevels of active ingredients in a therapeutic composition of theinvention can be varied so as to administer an amount of the activecompound(s) that is effective to achieve the desired therapeuticresponse for a particular subject. The selected dosage level will dependupon a variety of factors including the activity of the therapeuticcomposition, formulation, the route of administration, combination withother drugs or treatments, age, the age-related disease or condition,the degenerative disease, the function-decreasing disorder, thesymptoms, and the physical condition and prior medical history of thesubject being treated. In some embodiments, a minimal dose isadministered, and dose is escalated in the absence of dose-limitingtoxicity. Determination and adjustment of a therapeutically effectivedose, as well as evaluation of when and how to make such adjustments,are known to those of ordinary skill in the art of medicine.

The frequency of dosing may be daily or once, twice, three times or moreper week or per month, as needed as to effectively treat the symptoms.The timing of administration of the treatment relative to the diseaseitself and duration of treatment will be determined by the circumstancessurrounding the case. Treatment could begin immediately, such as at thesite of the injury as administered by emergency medical personnel.Treatment could begin in a hospital or clinic itself, or at a later timeafter discharge from the hospital or after being seen in an outpatientclinic. Duration of treatment could range from a single doseadministered on a one-time basis to a life-long course of therapeutictreatments.

Typical dosage levels can be determined and optimized using standardclinical techniques and will be dependent on the mode of administration.

(e) Subject

A subject may be a rodent, a human, a livestock animal, a companionanimal, or a zoological animal. In one embodiment, the subject may be arodent (e.g., a mouse, a rat, a guinea pig, etc.). In anotherembodiment, the subject may be a livestock animal. Non-limiting examplesof suitable livestock animals may include pigs, cows, horses, goats,sheep, llamas and alpacas. In still another embodiment, the subject maybe a companion animal. Non-limiting examples of companion animals mayinclude pets such as dogs, cats, rabbits, and birds. In yet anotherembodiment, the subject may be a zoological animal. As used herein, a“zoological animal” refers to an animal that may be found in a zoo. Suchanimals may include non-human primates, large cats, wolves, and bears.In a preferred embodiment, the subject is a human.

The human subject may be of any age. However, since senescent cells arenormally associated with aging, a human subject may be an older humansubject. In some embodiments, the human subject may be about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 years of age or older. Insome preferred embodiments, the human subject is 30 years of age orolder. In other preferred embodiments, the human subject is 40 years ofage or older. In other preferred embodiments, the human subject is 45years of age or older. In yet other preferred embodiments, the humansubject is 50 years of age or older. In still other preferredembodiments, the human subject is 55 years of age or older. In otherpreferred embodiments, the human subject is 60 years of age or older. Inyet other preferred embodiments, the human subject is 65 years of age orolder. In still other preferred embodiments, the human subject is 70years of age or older. In other preferred embodiments, the human subjectis 75 years of age or older. In still other preferred embodiments, thehuman subject is 80 years of age or older. In yet other preferredembodiments, the human subject is 85 years of age or older. In stillother preferred embodiments, the human subject is 90 years of age orolder.

Additionally, a subject in need thereof may be a subject suffering froman age-related disease or condition as described below.

(f) Aging and Age-Related Diseases

It has been demonstrated that senescent cells drive age-relatedpathologies and that selective elimination of these cells can prevent ordelay age-related deterioration. Thus, senescent cells may betherapeutic targets in the treatment of aging and age-related disease.As such, removal of senescent cells may delay tissue dysfunction andextend health span. Clearance of senescent cells is expected to improvetissue milieu, thereby improving the function of the remainingnon-senescent cells.

The present disclosure provides a method for delaying at least onefeature of aging in a subject, the method comprising administering acomposition comprising a therapeutically effective amount of a compoundof Formula (I) or a compound of Formula (II) to a subject. As usedherein, “a feature of aging” may include, but is not limited to,systemic decline of the immune system, muscle atrophy and decreasedmuscle strength, decreased skin elasticity, delayed wound healing,retinal atrophy, reduced lens transparency, reduced hearing,osteoporosis, sarcopenia, hair graying, skin wrinkling, poor vision,frailty, and cognitive impairment.

In an aspect, a composition of in the invention selectively killssenescent cells. In this way, targeting senescent cells during thecourse of aging may be a preventative strategy. Accordingly,administration of a composition comprising a therapeutically effectiveamount of a compound of Formula (I) or a compound of Formula (II) to asubject may prevent comorbidity and delay mortality in an older subject.Further, selective killing of senescent cells may boost the immunesystem, extend the health span, and improve the quality of life in asubject. Additionally, selective killing of senescent cells may delaysarcopenia. Sarcopenia is the degenerative loss of skeletal muscle mass,quality, and strength associated with aging. As such, a delay insarcopenia may reduce frailty, reduce risk of falling, reduce fractures,and reduce functional disability in a subject. Furthermore, selectivekilling of senescent cells may delay aging of the skin. Aged skin hasincreased wrinkles, decreased immune barrier function and increasedsusceptibility to skin cancer and trauma. As such, selective killing ofsenescent cells may delay skin wrinkling, delay the onset of decreasedimmune barrier function and decrease susceptibility to skin cancer andtrauma in a subject. Selective killing of senescent cells may also delaythe onset of retinal atrophy and reduced lens transparency as measuredby vision tests.

Methods of measuring aging are known in the art. For example, aging maybe measured in the bone by incident non-vertebral fractures, incidenthip fractures, incident total fractures, incident vertebral fractures,incident repeat fractures, functional recovery after fracture, bonemineral density decrease at the lumbar spine and hip, rate of kneebuckling, NSAID use, number of joints with pain, and osteoarthritis.Aging may also be measured in the muscle by functional decline, rate offalls, reaction time and grip strength, muscle mass decrease at upperand lower extremities, and dual tasking 10-meter gait speed. Further,aging may be measured in the cardiovascular system by systolic anddiastolic blood pressure change, incident hypertension, majorcardiovascular events such as myocardial infarction, stroke, congestiveheart disease, and cardiovascular mortality. Additionally, aging may bemeasured in the brain by cognitive decline, incident depression, andincident dementia. Also, aging may be measured in the immune system byrate of infection, rate of upper respiratory infections, rate offlu-like illness, incident severe infections that lead to hospitaladmission, incident cancer, rate of implant infections, and rate ofgastrointestinal infections. Other indications of aging may include, butnot limited to, decline in oral health, tooth loss, rate of GI symptoms,change in fasting glucose and/or insulin levels, body composition,decline in kidney function, quality of life, incident disabilityregarding activities of daily living, and incident nursing homeadmission. Methods of measuring skin aging are known in the art and mayinclude trans-epidermal water loss (TEWL), skin hydration, skinelasticity, area ratio analysis of crow's feet, sensitivity, radiance,roughness, spots, laxity, skin tone homogeneity, softness, and relief(variations in depth).

The present disclosure also provides a method of treating an age-relateddisease or condition, the method comprising administering a compositioncomprising a therapeutically effective amount of a compound of Formula(I) or a compound of Formula (II) to a subject in need thereof, providedthe age-related disease or condition is not cancer. As used herein,“age-related disease or condition” may include, but is not limited to, adegenerative disease or a function-decreasing disorder such asAlzheimer's disease, Parkinson's disease, cataracts, maculardegeneration, glaucoma, atherosclerosis, acute coronary syndrome,myocardial infarction, stroke, hypertension, idiopathic pulmonaryfibrosis (IPF), chronic obstructive pulmonary disease (COPD),osteoarthritis, type 2 diabetes, obesity, fat dysfunction, coronaryartery disease, cerebrovascular disease, periodontal disease, cancertreatment-related disability such as atrophy and fibrosis in varioustissues, brain and heart injury, and therapy-related myelodysplasticsyndromes, and diseases associated with accelerated aging and/or defectsin DNA damage repair and telomere maintenance such as progeroidsyndromes (i.e., Hutchinson-Gilford progeria syndrome, Werner syndrome,Bloom syndrome, Rothmund-Thomson Syndrome, Cockayne syndrome, xerodermapigmentosum, trichothiodystrophy, combined xerodermapigmentosum-Cockayne syndrome, restrictive dermopathy), ataxiatelangiectasia, Fanconi anemia, Friedreich's ataxia, dyskeratosiscongenital, aplastic anemia, IPF, and others. Methods of diagnosing andidentifying an age-related disease or condition are known in the art.

Definitions

Compounds useful in the compositions and methods include those describedherein in any of their pharmaceutically acceptable forms, includingisomers such as diastereomers and enantiomers, salts, solvates, andpolymorphs, as well as racemic mixtures and pure isomers of thecompounds described herein, where applicable.

The compounds described herein have asymmetric centers. Compounds of thepresent disclosure containing an asymmetrically substituted atom may beisolated in optically active or racemic form. All chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated.

When introducing elements of the embodiments described herein, thearticles “a”, “an”, “the” and “said” are intended to mean that there areone or more of the elements. The terms “comprising”, “including” and“having” are intended to be inclusive and mean that there may beadditional elements other than the listed elements.

“Bcl-2” as used herein alone or as part of a group references to amember of the Bcl-2 family of proteins comprise the following Bcl-x_(L),MCL-1, Bcl-W, BFL-1/A1, Bcl-B, BAX, BAK, and BOK.

“Alkyl” as used herein alone or as part of a group refers to saturatedmonovalent hydrocarbon radicals having straight or branched hydrocarbonchains or, in the event that at least 3 carbon atoms are present, cyclichydrocarbons or combinations thereof and contains 1 to 20 carbon atoms(C₁-C₂₀ alkyl), suitably 1 to 10 carbon atoms (C₁-C₁₀ alkyl), preferably1 to 8 carbon atoms (C₁-C₈ alkyl), more preferably 1 to 6 carbon atoms(C₁-C₄ alkyl), and even more preferably 1 to 4 carbon atoms (C₁-C₄alkyl). Examples of alkyl radicals include methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

“Aryl” as used herein, alone or as part of a group, includes an organicradical derived from an aromatic hydrocarbon by removal of one hydrogen,and includes monocyclic and polycyclic radicals, such as phenyl,biphenyl, naphthyl.

“Cycloalkyl” as used herein, alone or in combination, means a saturatedor partially saturated monocyclic, bicyclic or tricyclic alkyl radicalwherein each cyclic moiety contains from about 3 to about 8 carbonatoms, more preferably from about 3 to about 6 carbon atoms. Examples ofsuch cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like.

“Heteroatom” means an atom other than carbon e.g., in the ring of aheterocyclic group or the chain of a heterogeneous group. Preferably,heteroatoms are selected from the group consisting of sulfur,phosphorous, nitrogen, and oxygen atoms. Groups containing more than oneheteroatom may contain different heteroatoms.

“Heteroaryl” as used herein, along or in combination, includes anorganic radical derived from an aromatic hydrocarbon by removal of onehydrogen and includes at least one heteroatom. Examples of heteroarylincludes pyrrole, thiophene, furan, indole, pyrazine, pyridine,triazole, imidazole, thiazole, oxazole and the like.

“Substituted” means that one or more of the hydrogen atoms bonded tocarbon atoms in the chain or ring have been replaced with othersubstituents. Suitable substituents include monovalent hydrocarbongroups including alkyl groups such as methyl groups and monovalentheterogeneous groups including alkoxy groups such as methoxy groups.“Unsubstituted” means that the carbon chain or ring contains no othersubstituents other than carbon and hydrogen.

“Branched” means that the carbon chain is not simply a linear chain.“Unbranched” means that the carbon chain is a linear carbon chain.

“Heteroatom” means an atom other than carbon e.g., in the ring of aheterocyclic group or the chain of a heterogeneous group. Preferably,heteroatoms are selected from the group consisting of sulfur,phosphorous, nitrogen and oxygen atoms. Groups containing more than oneheteroatom may contain different heteroatoms.

“Heterocyclic group” means a saturated or unsaturated ring structurecontaining carbon atoms and 1 or more heteroatoms in the ring.Heterocyclic groups are not aromatic. Heterocyclic groups are monocyclicor polycyclic. Polycyclic heteroaromatic groups can be fused, spiro, orbridged ring systems. Monocyclic heterocyclic groups contain 4 to 10member atoms (i.e., including both carbon atoms and at least 1heteroatom), suitably 4 to 7, and more suitably 5 to 6 in the ring.Bicyclic heterocyclic groups contain 8 to 18 member atoms, suitably 9 or10 in the rings.

“Isomer”, “isomeric form”, “stereochemically isomeric forms” or“stereolsomeric forms”, as used herein, defines all possible isomeric aswell as conformational forms, made up of the same atoms bonded by thesame sequence of bonds but having different three-dimensional structureswhich are not interchangeable, which compounds or intermediates obtainedduring said process may possess. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms which saidcompound may possess. Said mixture may contain all diastereoisomers,epimers, enantiomers and/or conformers of the basic molecular structureof said compound. More in particular, stereogenic centers may have theR- or S-configuration, diastereoisomers may have a syn- oranti-configuration, substituents on bivalent cyclic saturated radicalsmay have either the cis- or trans-configuration and alkenyl radicals mayhave the E- or Z-configuration. All stereochemically isomeric forms ofsaid compound both in pure form or in admixture with each other areintended to be embraced within the scope of the present invention.

EXAMPLES

The following examples are included to demonstrate various embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

The compounds of the present invention may be prepared in a number ofways well known to one skilled in the art of organic synthesis. Morespecifically, the novel compounds of this invention may be preparedusing the reactions and techniques described herein. In the descriptionof the synthetic methods described below, it is to be understood thatall proposed reaction conditions, including choice of solvent, reactionatmosphere, reaction temperature, duration of the experiment and workupprocedures, are chosen to be the conditions standard for that reaction.It is understood by one skilled in the art of organic synthesis that thefunctionality present on various portions of the molecule must becompatible with the reagents and reactions proposed. Such restrictionsto the substituents, which are not compatible with the reactionconditions, will be apparent to one skilled in the art and alternatemethods must then be used. Unless otherwise stated, the startingmaterials for the examples contained herein are either commerciallyavailable or are readily prepared by standard methods from knownmaterials. The compounds of Formula (I) or Formula (II) may besynthesized through standard organic chemistry methodology andpurification known to those trained in the art of organic synthesis byusing commercially available starting materials and reagents.

The following abbreviations are used: s: singlet; d: doublet; t:triplet; q: quartet; m: multiplet; dd: doublet of doublet; dt: doubletof triplet: dq: doublet of quartet; br: broad; AcOH=acetic acid;DCM=dichloromethane; DIPEA=N,N-diisopropylethylamine;DMAP=4-dimethylaminopyridine; DMF=N,N-dimethylformamide;DMSO=dimethylsulfoxide;EDCI=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;EDTA=ethylenediaminetetraacetic acid; EtOAc=ethyl acetate; FBS=fetalbovine serum;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; HCl=hydrochloric acid; MOMCl=chloromethylmethyl ether; PBS=phosphate buffered saline; TBAF=tetra-n-butylammoniumfluoride; TBSCl=tert-butyldimethylchlorosilane; TBS-T=tris-bufferedsaline; TEA=triethylamine; THF=tetrahydrofuran; and TFA=trifluoroaceticacid.

Example 1: Synthesis of XZ-13906 Preparation of(4-bromo-2-fluorophenoxy)(tert-butyl)dimethylsilane (2)

4-Bromo-2-fluorophenol (1.0 g, 5.24 mmol), TBSCl (1.03 g, 6.83 mmol) andimidazole (713 mg, 10.48 mmol) were dissolved in 20 mL DMF and themixture was stirred at room temperature for 16 hours. Water was added tothe reaction mixture and extracted with EtOAc. The combined organicphases were washed with water x1, brine x1, dried over Na₂SO₄, filteredand evaporated to dryness. The resulting oil was further purified bycolumn chromatography to afford 1.60 g compound 2 as colorless oil.Yield 100%. ¹H NMR (400 MHz, CDCl₃) δ 7.22 (dd, J=10.1, 2.4 Hz, 1H),7.15-7.07 (m, 1H), 6.79 (t, J=8.7 Hz, 1H), 1.00 (s, 9H), 0.19 (d, J=0.9Hz, 6H) ppm.

Preparation of tert-butyl 4-(prop-2-ynyl)piperazine-1-carboxylate (4)

1-Boc-Piperazine 3 (1.0 g, 5.38 mmol), 80% 3-bromoprop-1-yne toluenesolution (900 μL, 8.07 mmol), and DIPEA (1.78 mL, 10.76 mmol) weredissolved in 25 mL DCM and the mixture was stirred at room temperaturefor 16 hours. Water was added to the reaction mixture and the aqueousphase was extracted with DCM. The combined organic phases were washedwith brine, dried over Na₂SO₄, filtered and evaporated to dryness. Theresulting oil was further purified by column chromatography to afford1.13 g compound 4. Yield 94%. ¹H NMR (400 MHz, CDCl₃) δ 3.54-3.42 (m,4H), 3.33 (d, J=2.4 Hz, 2H), 2.57-2.46 (m, 4H), 2.26 (t, J=2.4 Hz, 1H),1.46 (s, 9H) ppm.

Preparation of tert-butyl4-(3-(4-(tert-butyldimethylsilyloxy)-3-fluorophenyl)prop-2-ynyl)piperazine-1-carboxylate(5)

A mixture of compound 2 (612 mg, 2 mmol), compound 4 (448 mg, 2 mmol),Pd(PPh₃)₄ (68 mg, 0.06 mmol), CuI (12 mg, 0.06 mmol), and TEA (700 μL,4.2 mmol) were stirred in 15 mL DMF at 100° C. under Argon for 20 hours.Water was added to the reaction mixture and extracted with EtOAc. Thecombined organic phases were washed with water x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The resulting oil wasfurther purified by column chromatography to afford 220 mg compound 5.Yield 25%. ¹H NMR (400 MHz, CDCl₃) δ 7.13 (dd, J=11.1, 2.0 Hz, 1H),7.10-7.04 (m, 1H), 6.83 (t, J=8.5 Hz, 1H), 3.58-3.41 (m, 6H), 2.68-2.50(m, 4H), 1.47 (s, 9H), 1.00 (s, 9H), 0.19 (d, J=0.9 Hz, 6H) ppm.

Preparation of tert-butyl4-(3-(3-fluoro-4-hydroxyphenyl)prop-2-ynyl)piperazine-1-carboxylate (6)

To a solution of compound 5 (180 mg, 0.4 mmol) in 5 mL THF was added 0.8mL TBAF solution (1.0 M in THF) dropwise. After 30 minutes, water wasadded to the reaction mixture and extracted with EtOAc. The organicphase was washed with saturated NH₄Cl (aq) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The resulting mixture wasfurther purified by column chromatography to afford 126 mg compound 6 asbrown solid. Yield 94%. ¹H NMR (400 MHz, CDCl₃) δ 7.09-7.00 (m, 2H),6.89 (t, J=8.8 Hz, 1H), 3.59-3.45 (m, 6H), 2.70-2.54 (m, 4H), 2.08 (s,1H), 1.47 (s, 9H) ppm.

Preparation of2-(2,6-dioxopiperidin-3-yl)-4-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethylamino) isoindoline-1,3-dione (9)

2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (7) wassynthesized according to reported method with minor modifications (Chem.Biol. 22:755-763, 2015). Compound 7 (100 mg, 0.36 mmol), amine 8 (68 mg,0.36 mmol), and DIPEA (120 μL, 0.72 mmol) in 4 mL DMF were stirred at90° C. for 16 hours. Water was added to the reaction mixture andextracted with EtOAc. The organic phase was washed with water x1, brinex1, dried over Na₂SO₄, filtered and evaporated to dryness. The resultingmixture was further purified by column chromatography to afford 95 mgcompound 9 as a green solid. Yield 59%. ¹H NMR (400 MHz, CDCl₃) δ 8.02(s, 1H), 7.64-7.34 (m, 1H), 7.10 (d, J=7.1 Hz, 1H), 6.93 (d, J=8.6 Hz,1H), 6.67-6.11 (m, 1H), 4.91 (dd, J=12.1, 5.3 Hz, 1H), 4.20 (d, J=2.2Hz, 2H), 3.83-3.60 (m, 10H), 3.55-3.40 (m, 2H), 2.99-2.60 (m, 3H), 2.43(t, J=2.1 Hz, 1H), 2.21-2.03 (m, 1H) ppm. YesCL Preparation of2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(3-(4-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)prop-1-ynyl)-2-fluorophenoxy)propyl)thiazole-4-carboxylicacid (11)

Compound 10 was synthesized according to reported method with minormodifications (ACS Med Chem Lett. 5:1088-1093, 2014). Compound 6 (200mg, 0.60 mmol) in 5 mL DMF was cooled to 0° C. and 40 mg 95% NaH wasadded to the solution. The resulting reaction mixture was stirred for 10min before the addition of compound 10 (250 mg, 0.40 mmol) in 5 mL THF.The mixture was stirred at room temperature for 3 hours and quenched byadding 1 mL water. The pH was adjusted to 5 using 1N HCl (aq) and theresulted solution was extracted with EtOAc. The organic phase was washedwith water x1, brine x1, dried over Na₂SO₄, filtered and evaporated todryness. The resulting mixture was further purified by columnchromatography to afford 130 mg compound 11. Yield 41%. ¹H NMR (400 MHz,CDCl₃) δ 7.90-7.76 (m, 1H), 7.69-7.59 (m, 1H), 7.54-7.41 (m, 1H),7.36-7.29 (m, 4H), 7.14-7.05 (m, 2H), 6.80 (t, J=8.5 Hz, 1H), 4.93 (s,2H), 4.00 (t, J=6.2 Hz, 2H), 3.79-3.65 (m, 2H), 3.57-3.49 (m, 6H), 3.28(t, J=7.3 Hz, 2H), 3.09-2.88 (m, 2H), 2.74-2.46 (m, 4H), 2.30-2.06 (m,2H), 1.46 (s, 9H) ppm.

Preparation of2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(3-(2-fluoro-4-(3-(piperazin-1-yl)prop-1-ynyl)phenoxy)propyl)thiazole-4-carboxylicacid (12)

A mixture of compound 11 (130 mg) and TFA (1 mL) in 3 mL DCM was stirredat room temperature for 1 hour. The solvent was removed under reducedpressure and the crude product was crystallized in Et₂O and MeOH to give110 mg compound 12 as a pale yellow solid. ¹H NMR (400 MHz, CD₃OD) δ7.93 (d, J=7.7 Hz, 1H), 7.78 (d, J=7.7 Hz, 1H), 7.63 (d, J=7.2 Hz, 1H),7.52-7.28 (m, 5H), 7.18-7.07 (m, 2H), 6.99 (t, J=8.7 Hz, 1H), 4.91 (s,2H), 4.07 (t, J=6.1 Hz, 2H), 3.89-3.77 (m, 2H), 3.62 (s, 2H), 3.28-3.20(m, 6H), 3.09-3.05 (m, 2H), 2.93-2.80 (m, 4H), 2.20-2.07 (m, 2H) ppm.

Preparation of5-(3-(4-(3-(4-(4-azidobutanoyl)piperazin-1-yl)prop-1-ynyl)-2-fluorophenoxy)propyl)-2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)thiazole-4-carboxylicacid (13)

Compound 12 (100 mg) and TEA (157 μL) in 4 mL DCM was stirred at roomtemperature. 4-Azidobutanoyl chloride (16.4 mg) in 660 μL DCM was thenadded dropwise to the mixture. The reaction was quenched after stirredfor 10 minutes by adding 1 mL MeOH. DCM was added and the mixture waswashed water x1, brine x1, dried over Na₂SO₄, filtered and evaporated todryness. The crude product was crystallized in MeOH to give 85 mg paleyellow solid. ¹H NMR (400 MHz, CDCl₃) δ 7.86 (d, J=7.8 Hz, 1H),7.69-7.59 (m, 2H), 7.44-7.29 (m, 4H), 7.15-7.05 (m, 2H), 6.82 (t, J=8.7Hz, 1H), 4.95 (s, 2H), 4.04 (t, J=6.3 Hz, 2H), 3.81-3.64 (m, 6H),3.44-3.24 (m, 6H), 3.06 (t, J=5.9 Hz, 2H), 2.89-2.58 (m, 4H), 2.42 (t,J=7.2 Hz, 2H), 2.22-2.11 (m, 2H), 1.99-1.87 (m, 2H) ppm.

Preparation of2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(3-(4-(3-(4-(4-(4-((2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)prop-1-ynyl)-2-fluorophenoxy)propyl)thiazole-4-carboxylicacid (XZ-13906)

To a mixture of compound 13 (18 mg), compound 9 (10 mg) in 1 mL t-BuOHunder Argon was added CuSO₄.5H₂O (1.0 mg) and sodium ascorbate (0.8 mg)in 0.2 mL water. The mixture was stirred at 65° C. for 16 hours andextracted with DCM. The organic phase was washed brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified using reverse phase preparative HPLC to give 4.0 mg pureproduct as yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.17 (s, 1H),7.92-7.82 (m, 2H), 7.71 (d, J=6.9 Hz, 1H), 7.53 (s, 1H), 7.50-7.43 (m,2H), 7.37 (t, J=7.5 Hz, 1H), 7.33-7.27 (m, 2H), 7.15-7.02 (m, 3H), 6.87(d, J=8.6 Hz, 1H), 6.79 (t, J=8.4 Hz, 1H), 6.48 (br s, 1H), 4.99-4.83(m, 3H), 4.70-4.53 (m, 2H), 4.37 (t, J=5.9 Hz, 2H), 4.11-3.94 (m, 4H),3.82-3.56 (m, 16H), 3.42 (t, J=4.8 Hz, 2H), 3.35-3.06 (m, 6H), 3.02 (t,J=5.7 Hz, 2H), 2.89-2.67 (m, 3H), 2.33-2.05 (m, 7H) ppm.

Example 2: Synthesis of XZ-13942

Preparation of ethyl5-(3-azidopropyl)-2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)thiazole-4-carboxylate(14)

Compound 10 (100 mg) and NaN₃ (13 mg) were stirred in 5 mL DMSO at 45°C. overnight. The mixture was poured into water and extracted withEtOAc. The organic phase was washed with water x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness to give 85 mg pure product aswhite solid. Yield 98%. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J=6.7 Hz,1H), 7.54 (d, J=7.6 Hz, 1H), 7.35-7.26 (m, 4H), 7.18 (t, J=7.6 Hz, 1H),4.87 (s, 2H), 4.28 (q, J=7.1 Hz, 2H), 3.90-3.79 (m, 2H), 3.31 (t, J=6.6Hz, 2H), 3.11 (t, J=7.4 Hz, 2H), 3.03-2.92 (m, 2H), 1.98-1.82 (m, 2H),1.31 (t, J=7.1 Hz, 3H) ppm.

Preparation of methoxymethyl5-(3-azidopropyl)-2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)thiazole-4-carboxylate(15)

Compound 14 (85 mg) and NaOH (26.5 mg) were stirred in a mixture ofethanol and water at 50° C. for 5 hours. The mixture was cooled to roomtemperature and neutralized with 1N HCl (aq.). The precipitated solidwas collected and dissolved in 4 mL DMF. Then Na₂CO₃ (17 mg) and MOMCl(12 mg) was added into the mixture. After 16 hours, the mixture waspoured into water and extracted with EtOAc. The organic phase was washedwith water x1, brine x1, dried over Na₂SO₄, filtered and evaporated todryness. The resulting mixture was purified via column chromatographyusing EtOAc and hexanes as eluents to afford 53 mg compound 15. Yield61%. ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J=7.7 Hz, 1H), 7.75 (d, J=7.1Hz, 1H), 7.68 (d, J=7.9 Hz, 1H), 7.51-7.29 (m, 5H), 5.40 (s, 2H), 4.94(s, 2H), 3.90 (t, J=6.1 Hz, 2H), 3.50 (s, 3H), 3.35 (t, J=6.8 Hz, 2H),3.17 (t, J=7.5 Hz, 2H), 3.08 (t, J=6.1 Hz, 2H), 1.99-1.88 (m, 2H) ppm.

Preparation of2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(3-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)thiazole-4-carboxylicacid (XZ13942)

To a mixture of compound 15 (10 mg), compound 9 (8.7 mg) in 2 mLt-BuOH-THF (1:1, v/v) under argon was added CuSO₄.5H₂O (0.9 mg) andsodium ascorbate (0.7 mg) in 0.4 mL water. The mixture was stirred at60° C. for 16 hours and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 14 mg compound 16. Yield 78%. Compound 16(9.0 mg) and 0.1 mL TFA was stirred in 3 mL DCM for 1 hour. The solventwas removed under reduced pressure. Then Et₂O was added into the residueand the precipitated solid was collected to afford 8.4 mg pure XZ13942.Yield 79%. ¹H NMR (400 MHz, CDCl₃) δ 10.03 (s, 1H), 7.94 (d, J=6.4 Hz,1H), 7.87 (d, J=7.7 Hz, 1H), 7.77 (d, J=6.7 Hz, 1H), 7.64-7.29 (m, 6H),7.07 (d, J=7.0 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H), 5.03-4.86 (m, 3H),4.75-4.55 (m, 2H), 4.46-4.30 (m, 2H), 3.86-3.58 (m, 12H), 3.46-3.33 (m,2H), 3.25-2.98 (m, 4H), 2.92-2.68 (m, 3H), 2.28-2.05 (m, 3H) ppm.

Example 3: Synthesis of XZ-14424

Preparation of2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)isoindoline-1,3-dione (18)

Compound 7 (107 mg), amine 17 (84 mg), and DIPEA (193 μL) in 5 mL DMFwere stirred at 85° C. for 16 hours. Water was added to the reactionmixture and extracted with EtOAc. The organic phase was washed withwater x1, brine x1, dried over Na₂SO₄, filtered and evaporated todryness. The resulting mixture was purified by column chromatographyusing EtOAc and hexanes as eluents to afford 50 mg compound 18 as agreen solid. Yield 32%. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (s, 1H),7.62-7.35 (m, 1H), 7.11 (d, J=7.1 Hz, 1H), 6.93 (d, J=8.5 Hz, 1H), 4.92(dd, J=11.9, 5.3 Hz, 1H), 4.21 (d, J=2.3 Hz, 2H), 3.78-3.66 (m, 6H),3.49 (t, J=5.4 Hz, 2H), 2.93-2.68 (m, 3H), 2.48-2.41 (m, 1H), 2.18-2.09(m, 1H) ppm.

Preparation of methoxymethyl5-(3-(4-(3-(4-(4-azidobutanoyl)piperazin-1-yl)prop-1-yn-1-yl)-2-fluorophenoxy)propyl)-2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)thiazole-4-carboxylate(19)

Compound 13 (26 mg), Na₂CO₃ (4.1 mg) and MOMCl (2.8 mg) were stirred in2 mL DMF for 24 hours. Then it was poured into water and extracted withEtOAc. The organic phase was washed with water x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The resulting mixture waspurified via column chromatography using DCM and methanol as eluents toafford 15 mg compound 19. Yield 58%. ¹H NMR (400 MHz, CDCl₃) δ 7.90-7.77(m, 1H), 7.54 (d, J=7.6 Hz, 1H), 7.37-7.25 (m, 4H), 7.18 (t, J=7.6 Hz,1H), 7.12-7.04 (m, 2H), 6.81 (t, J=8.4 Hz, 1H), 5.34 (s, 2H), 4.88 (s,2H), 4.03 (t, J=6.2 Hz, 2H), 3.81 (t, J=6.0 Hz, 2H), 3.76-3.64 (m, 2H),3.63-3.49 (m, 4H), 3.44 (s, 3H), 3.36 (t, J=6.3 Hz, 2H), 3.25 (t, J=7.4Hz, 2H), 3.00 (t, J=5.9 Hz, 2H), 2.71-2.53 (m, 4H), 2.40 (t, J=7.2 Hz,2H), 2.23-2.06 (m, 2H), 1.98-1.84 (m, 2H) ppm.

Preparation of2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(3-(4-(3-(4-(4-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)prop-1-yn-1-yl)-2-fluorophenoxy)propyl)thiazole-4-carboxylicacid (XZ14424)

To a mixture of compound 19 (13.0 mg), compound 18 (8.0 mg) in 2 mLt-BuOH-THF (1:1, v/v) under argon was added CuSO₄.5H₂O (0.82 mg) andsodium ascorbate (0.65 mg) in 0.4 mL water. The mixture was stirred at55° C. for 16 hours and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 20 mg compound 20. Yield 91%. Compound 20(20.0 mg) and 0.1 mL HCl solution (4.0 M in 1,4-dioxane) was stirred in4 mL DCM-methanol (3:1, v/v) for 3 hours. The solvents were removedunder reduced pressure. Et₂O was then added to the residue and theprecipitated solid was collected to afford 15.4 mg pure XZ14424. Yield73%. ¹H NMR (400 MHz, CD₃OD) δ 8.02 (s, 1H), 7.92 (d, J=7.9 Hz, 1H),7.79 (d, J=7.9 Hz, 2H), 7.61-7.44 (m, 4H), 7.36 (t, J=7.5 Hz, 1H),7.31-7.18 (m, 2H), 7.12-6.95 (m, 3H), 5.14 (s, 2H), 5.01 (dd, J=12.7,5.4 Hz, 1H), 4.64 (s, 2H), 4.47 (t, J=6.5 Hz, 2H), 4.34 (s, 2H), 4.16(t, J=5.5 Hz, 2H), 3.99-3.88 (m, 2H), 3.77-3.44 (m, 14H), 3.38-3.33 (m,4H), 3.26-3.19 (m, 2H), 2.89-2.62 (m, 3H), 2.52-2.38 (m, 2H), 2.25-2.04(m, 5H) ppm.

Example 4: Synthesis of XZ-14418

Preparation ofN-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)acetamide(22)

Lenalidomide (61 mg), compound 21 (57 mg), HATU (94 mg) and DIPEA (59μL) were stirred in 5 mL DCM overnight. The mixture was concentratedunder reduced pressure and purified via column chromatography using DCMand methanol as eluents to afford 58 mg compound 22. Yield 56%. ¹H NMR(400 MHz, CDCl₃) δ 8.92 (s, 1H), 7.97 (s, 1H), 7.74 (d, J=7.5 Hz, 1H),7.68 (d, J=7.9 Hz, 1H), 7.49 (t, J=7.7 Hz, 1H), 5.20 (dd, J=13.3, 5.1Hz, 1H), 4.45 (s, 2H), 4.14 (d, J=3.4 Hz, 2H), 3.96 (s, 2H), 3.83-3.57(m, 8H), 2.98-2.70 (m, 2H), 2.49-2.28 (m, 2H), 2.27-2.13 (m, 1H) ppm.

Preparation of2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(3-(4-(3-(4-(4-(4-((2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-ylamino)-2-oxoethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)prop-1-ynyl)-2-fluorophenoxy)propyl)thiazole-4-carboxylicacid (XZ14418)

To a mixture of compound 19 (12.0 mg), compound 22 (7.4 mg) in 4 mLt-BuOH-THF (1:3, v/v) under Argon was added CuSO₄.5H₂O (0.70 mg) andsodium ascorbate (0.56 mg) in 0.4 mL water. The mixture was stirred at55° C. for 16 hours and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 15.0 mg compound 23. Yield 85%. Compound23 (15.0 mg) and 0.1 mL HCl solution (4.0 M in 1,4-dioxane) was stirredin 4 mL 5 mL DCM for 10 minutes. The solvent was removed under reducedpressure. Then Et₂O was added into the residue and the precipitatedsolid was collected to afford 11.8 mg pure XZ14418. Yield 75%. ¹H NMR(400 MHz, CD₃OD) δ 8.00-7.89 (m, 2H), 7.84-7.76 (m, 2H), 7.69 (d, J=7.8Hz, 1H), 7.64 (d, J=7.1 Hz, 1H), 7.59-7.43 (m, 4H), 7.37 (t, J=7.6 Hz,1H), 7.31-7.19 (m, 2H), 7.06 (t, J=8.5 Hz, 1H), 5.23-5.04 (m, 3H),4.57-4.45 (m, 4H), 4.41 (t, J=6.8 Hz, 2H), 4.33 (s, 2H), 4.23-4.12 (m,4H), 3.93 (t, J=5.7 Hz, 2H), 3.82-3.63 (m, 10H), 3.37-3.33 (m, 8H), 3.21(t, J=5.5 Hz, 2H), 2.96-2.67 (m, 2H), 2.57-2.36 (m, 3H), 2.28-2.11 (m,5H) ppm.

Example 5: Synthesis of XZ-14455

Preparation of(2S,4R)-1-((S)-2-tert-butyl-4-oxo-6,9,12-trioxa-3-azapentadec-14-yne)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(25)

A mixture of compound 21, compound 24, HATU, and DIPEA in DCM wasstirred at room temperature overnight. The mixture was concentratedunder reduced pressure and purified via column chromatography using DCMand methanol as eluents to afford the title compound. ¹H NMR (400 MHz,CDCl₃) δ 8.68 (s, 1H), 7.60-7.25 (m, 6H), 4.70 (t, J=8.0 Hz, 1H),4.62-4.37 (m, 3H), 4.33 (dd, J=15.0, 5.3 Hz, 1H), 4.22-4.10 (m, 2H),4.02-3.91 (m, 3H), 3.65-3.46 (m, 9H), 2.55-2.37 (m, 5H), 2.21-2.09 (m,1H), 0.91 (s, 9H) ppm.

Preparation of2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(3-(2-fluoro-4-(3-(4-(4-(4-((S)-12-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-13,13-dimethyl-10-oxo-2,5,8-trioxa-11-azatetradecyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)prop-1-yn-1-yl)phenoxy)propyl)thiazole-4-carboxylicacid (XZ14455)

To a mixture of compound 19 (17.0 mg), compound 25 (17.0 mg) in 4 mLt-BuOH-THF (1:1, v/v) under argon was added CuSO₄.5H₂O (1.0 mg) andsodium ascorbate (0.8 mg) in 0.4 mL water. The mixture was stirred at50° C. for 5 hours and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 14.9 mg compound 26. Yield 51%. Compound26 (3.5 mg) and 0.1 mL TFA was stirred in 2 mL DCM for 6 hours. Thesolvent was removed under reduced pressure. Then Et₂O was added into theresidue. The precipitated solid was filtered and washed with EtOAcfollowed by DCM-hexanes (1:1 v/v) to afford 3.5 mg pure XZ14455. Yield83%. ¹H NMR (400 MHz, CD₃OD) δ 8.86 (s, 1H), 8.00-7.89 (m, 2H), 7.78 (d,J=7.9 Hz, 1H), 7.71-7.59 (m, 1H), 7.53-7.30 (m, 8H), 7.29-7.17 (m, 2H),7.02 (t, J=8.5 Hz, 1H), 4.89 (s, 2H), 4.73-4.20 (m, 12H), 4.17-3.96 (m,4H), 3.92-3.59 (m, 15H), 3.40-3.33 (m, 2H), 3.28-3.23 (m, 2H), 3.07 (t,J=5.8 Hz, 2H), 2.49-2.36 (m, 5H), 2.31-1.94 (m, 6H), 1.02 (s, 9H) ppm.

Example 6: Synthesis of XZ-14439

Preparation of 2,2,2-trichloroethyl(R)-4-(3-((tert-butoxycarbonyl)amino)-4-(phenylthio)butyl)piperazine-1-carboxylate(29)

To a mixture of compound 27 (592 mg), compound 28 (753 mg), and TEA(1.12 mL) in 15 mL DCM was added 638 mg NaBH(OAc)₃. The solution wasstirred at room temperature for 1 hour. Then it was poured into waterand extracted with DCM. The organic phase was washed with brine x1,dried over Na₂SO₄, filtered and evaporated to dryness. The crude productwas purified via column chromatography using EtOAc and hexanes aseluents to afford 733 mg compound 29. Yield 68%. ¹H NMR (400 MHz, CDCl₃)δ 7.43-7.36 (m, 2H), 7.32-7.27 (m, 2H), 7.19 (t, J=7.3 Hz, 1H), 5.44 (brs, 1H), 4.76 (s, 2H), 3.99-3.84 (m, 1H), 3.72-3.49 (m, 4H), 3.23 (dd,J=13.3, 4.6 Hz, 1H), 3.10-2.95 (m, 1H), 2.61-2.31 (m, 6H), 1.96-1.61 (m,2H), 1.43 (s, 9H) ppm.

Preparation of 2,2,2-trichloroethyl(R)-4-(3-amino-4-(phenylthio)butyl)piperazine-1-carboxylate (30)

To a mixture of compound 29 (733 mg) in 5 mL DCM was added 5 mL HClsolution (4.0 M in 1,4-dioxane). The mixture was stirred at roomtemperature for 1 hour and the solvents were removed under reducedpressure. The solid was washed with Et₂O to afford 647 mg compound 30 aswhite solid. Yield 99%. ¹H NMR (400 MHz, CDCl₃) δ 7.41-7.33 (m, 2H),7.31-7.26 (m, 2H), 7.23-7.15 (m, 1H), 4.74 (s, 2H), 3.73-3.41 (m, 4H),3.20-2.66 (m, 5H), 2.58-2.28 (m, 6H), 1.84-1.57 (m, 2H) ppm.

Preparation of 2,2,2-trichloroethyl(R)-4-(4-(phenylthio)-3-((4-sulfamoyl-2-((trifluoromethyl)sulfonyl)phenyl)amino)butyl)piperazine-1-carboxylate(32)

A mixture of compound 30 (647 mg),4-fluoro-3-((trifluoromethyl)sulfonyl)benzenesulfonamide 31 (417 mg) andTEA (945 μL) in 20 mL acetonitrile was refluxed for 4 hours. The solventwas evaporated under reduced pressure and the crude product was purifiedvia column chromatography using EtOAc and hexanes as eluents to afford780 mg compound 32 as white solid. Yield 79%. ¹H NMR (400 MHz, CDCl₃) δ8.24 (d, J=2.2 Hz, 1H), 7.84 (d, J=9.1 Hz, 1H), 7.42-7.37 (m, 2H),7.36-7.27 (m, 3H), 7.05 (d, J=8.6 Hz, 1H), 6.65 (br s, 1H), 5.13 (br s,J=10.8 Hz, 2H), 4.76 (s, 2H), 3.94 (s, 1H), 3.58 (s, 4H), 3.16-2.97 (m,2H), 2.82-2.26 (m, 6H), 2.17 (s, 1H), 1.77 (s, 1H) ppm.

Preparation of 2,2,2-trichloroethyl(R)-4-(3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazine-1-carboxylate(34)

A mixture of compound 32 (780 mg),4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoicacid 33 (470 mg), EDCl (411 mg) and DMAP (262 mg) in DCM was stirred atroom temperature overnight. The solvent was evaporated under reducedpressure and the crude product was purified via column chromatographyusing DCM and methanol as eluents to afford 859 mg compound 34 as whitesolid. Yield 70%. ¹H NMR (400 MHz, CDCl₃) δ 8.35 (s, 1H), 8.10 (d, J=8.7Hz, 1H), 7.66 (d, J=8.0 Hz, 2H), 7.43-7.18 (m, 7H), 7.12-6.96 (m, 3H),6.74 (s, 1H), 6.56 (d, J=7.9 Hz, 1H), 4.74 (s, 2H), 3.93-3.83 (m, 1H),3.61-3.42 (m, 4H), 3.39-3.25 (m, 4H), 3.16-2.83 (m, 4H), 2.44-2.02 (m,15H), 1.77-1.60 (m, 1H), 1.56-1.42 (m, 2H), 0.98 (s, 6H) ppm.

Preparation of(R)-4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((1-(phenylthio)-4-(piperazin-1-yl)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(35)

Zinc powder (960 mg) was added to a mixture of compound 34 (316 mg) andAcOH (600 μL) in 20 mL THF. The reaction was stirred at room temperaturefor 5 hours. The solid was removed by filtration and the filtrate waspoured into water and extracted with EtOAc. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM,methanol, and TEA as eluents to afford 210 mg compound 35. Yield 78%. ¹HNMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.93 (d, J=9.2 Hz, 1H), 7.85 (d,J=8.6 Hz, 2H), 7.33-7.24 (m, 2H), 7.22-7.15 (m, 6H), 7.15-7.08 (m, 1H),6.92 (d, J=8.3 Hz, 2H), 6.77 (d, J=8.4 Hz, 1H), 6.66 (d, J=8.7 Hz, 2H),6.46 (d, J=9.3 Hz, 1H), 3.83-3.67 (m, 1H), 3.17-3.08 (m, 4H), 3.02-2.92(m, 5H), 2.89-2.78 (m, 1H), 2.72 (s, 2H), 2.64-2.13 (m, 12H), 2.04-1.91(m, 3H), 1.62-1.49 (m, 1H), 1.39 (t, J=6.3 Hz, 2H), 0.91 (s, 6H) ppm.

Preparation of(R)—N-((4-((4-(4-(4-azidobutanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)-4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzamide(36)

HATU (30 mg) was added to a mixture of compound 35 (50 mg),4-azidobutanoic acid (6.7 mg), DIPEA (13.5 μL) in 2 mL DCM. The mixturewas stirred at room temperature for 1 hour. The solvent was removedunder reduced pressure and the crude product was purified via columnchromatography using DCM and methanol as eluents to afford 40 mgcompound 36. Yield 72%. ¹H NMR (400 MHz, CDCl₃) δ 8.35 (d, J=2.2 Hz,1H), 8.11 (dd, J=9.2, 2.2 Hz, 1H), 7.67 (d, J=8.9 Hz, 2H), 7.40-7.35 (m,2H), 7.34-7.27 (m, 3H), 7.26-7.24 (m, 2H), 7.09 (d, J=8.5 Hz, 1H),7.02-6.96 (m, 2H), 6.76 (d, J=9.0 Hz, 2H), 6.58 (d, J=9.4 Hz, 1H),3.99-3.81 (m, 1H), 3.72-3.60 (m, 1H), 3.53-3.33 (m, 5H), 3.32-3.22 (m,4H), 3.11 (dd, J=13.8, 4.9 Hz, 1H), 3.00 (dd, J=13.8, 7.5 Hz, 1H), 2.87(s, 2H), 2.51-2.20 (m, 14H), 2.19-2.08 (m, 1H), 2.06-1.99 (m, 2H),1.97-1.85 (m, 2H), 1.71-1.64 (m, 1H), 1.46 (t, J=6.4 Hz, 2H), 0.97 (s,6H) ppm.

Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(4-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(XZ-14439)

To a mixture of compound 36 (7.5 mg), compound 9 (3.7 mg) in 2 mLt-BuOH-THF (1:1, v/v) under argon was added CuSO₄.5H₂O (0.35 mg) andsodium ascorbate (0.28 mg) in 0.4 mL water. The mixture was stirred at50° C. for 3 hours and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 5.9 mg XZ14439. Yield 56%. ¹H NMR (400MHz, CDCl₃ and CD₃OD) δ 8.33 (s, 1H), 8.10 (d, J=7.8 Hz, 1H), 7.79-7.65(m, 3H), 7.54-7.44 (m, 1H), 7.43-7.35 (m, 2H), 7.33-7.32 (m, 1H),7.30-7.22 (m, 4H), 7.12-7.02 (m, 2H), 6.99 (d, J=8.3 Hz, 2H), 6.92 (d,J=8.6 Hz, 1H), 6.77 (d, J=8.9 Hz, 2H), 6.61 (d, J=9.3 Hz, 1H), 5.02-4.85(m, 1H), 4.66 (s, 2H), 4.41 (t, J=6.6 Hz, 2H), 4.00-3.79 (m, 1H),3.80-3.58 (m, 12H), 3.52-3.38 (m, 4H), 3.31-3.23 (m, 4H), 3.12 (dd,J=13.8, 5.0 Hz, 1H), 3.02 (dd, J=13.9, 7.3 Hz, 1H), 2.84-2.77 (m, 5H),2.50-2.06 (m, 18H), 2.01 (s, 2H), 1.74-1.63 (m, 1H), 1.46 (t, J=6.4 Hz,2H), 0.98 (s, 6H) ppm.

Example 7: Synthesis of PZ-15227 Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(4-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(PZ-15227)

To a mixture of compound 36 (25.0 mg), compound 18 (11.0 mg) in 3 mLt-BuOH-THF (1:2, v/v) under Argon was added CuSO₄.5H₂O (1.15 mg) andsodium ascorbate (0.91 mg) in 0.3 mL water. The mixture was stirred at50° C. overnight and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 23 mg PZ15227. Yield 67%. ¹H NMR (400 MHz,CDCl₃ and CD₃OD) δ 9.05 (br s, 1H), 8.36 (s, 1H), 8.10 (d, J=7.8 Hz,1H), 7.79-7.64 (m, 3H), 7.54-7.42 (m, 1H), 7.43-7.22 (m, 7H), 7.10-7.02(m, 2H), 6.99 (d, J=7.2 Hz, 2H), 6.92 (d, J=8.8 Hz, 1H), 6.77 (d, J=8.8Hz, 2H), 6.61 (d, J=9.3 Hz, 1H), 6.50 (br s, 1H), 4.99-4.85 (m, 1H),4.69 (s, 2H), 4.42-4.37 (m, 2H), 4.00-3.77 (m, 1H), 3.80-3.58 (m, 8H),3.52-3.20 (m, 8H), 3.12-3.00 (m, 2H), 2.84-2.75 (m, 5H), 2.45-1.98 (m,20H), 1.74-1.60 (m, 1H), 1.46 (t, J=6.4 Hz, 2H), 0.97 (s, 6H) ppm.

Example 8: Synthesis of XZ-14509

Preparation of4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2-oxopiperidin-3-yl)isoindoline-1,3-dione(39)

Compound 7 (200 mg), amine 37 (178 mg), and DIPEA (240 μL) in 5 mL DMFwere stirred at 90° C. for 16 hours. Water was added to the reactionmixture and extracted with EtOAc. The organic phase was washed withwater x1, brine x1, dried over Na₂SO₄, filtered and evaporated todryness. The resulting mixture was purified by column chromatography toafford 183 mg compound 38. Yield 50%. To a mixture of compound 38 (160mg) in 5 mL DCM was added 0.5 mL TFA. The mixture was stirred at roomtemperature for 2 h and the solvent was evaporated under reducedpressure. The salt was washed with Et₂O to afford pure compound 39. ¹HNMR (400 MHz, CDCl₃) δ 9.63 (br s, 1H), 7.82 (br s, 2H), 7.48 (dd,J=8.4, 7.2 Hz, 1H), 7.04 (d, J=7.0 Hz, 1H), 6.87 (d, J=8.5 Hz, 1H),5.13-4.82 (m, 1H), 3.85-3.60 (m, 8H), 3.55-3.37 (m, 2H), 3.28-3.12 (m,2H), 2.81-2.58 (m, 3H), 2.09-1.88 (m, 1H) ppm.

Preparation of4-((R)-3-(4-(N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)-N-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)ethyl)piperazine-1-carboxamide(XZ-14509)

A mixture of compound 39 (20 mg), carbonyldiimidazole (CDI) (10 mg) andTEA (7.0 μL) in 3 mL DCM was stirred at room temperature for 2 hours.Compound 35 (15 mg) and DIPEA (0.05 mL) were then added into the abovesolution. The mixture was stirred overnight and quenched by the additionof NH₄Cl (aq.), extracted with DCM and the organic phase was washed withwater x1, brine x1, dried over Na₂SO₄, filtered and evaporated todryness. The crude product was purified by column chromatography usingDCM and methanol as eluents to afford 6.7 mg compound XZ14509. Yield31%. ¹H NMR (400 MHz, CDCl₃) δ 9.24 (br s, 1H), 8.35 (s, 1H), 8.14-7.98(m, 1H), 7.81-7.62 (m, 2H), 7.52-7.40 (m, 1H), 7.39-7.27 (m, 4H),7.24-7.15 (m, 1H), 7.12-6.94 (m, 4H), 6.87 (d, J=8.6 Hz, 1H), 6.73 (d,J=7.2 Hz, 2H), 6.66-6.57 (m, 1H), 6.56-6.46 (m, 1H), 5.20-5.02 (br s,1H), 5.00-4.83 (m, 1H), 3.95-3.81 (m, 1H), 3.75-3.69 (m, 2H), 3.67-3.61(m, 4H), 3.61-3.53 (m, 2H), 3.49-3.38 (m, 4H), 3.38-3.18 (m, 8H),3.12-2.95 (m, 2H), 2.88-2.66 (m, 5H), 2.47-2.18 (m, 12H), 2.17-1.98 (m,4H), 1.69-1.57 (m, 1H), 1.46 (t, J=6.3 Hz, 2H), 0.97 (s, 6H) ppm.

Example 9: Synthesis of XZ-14516 Preparation of4-(4-((2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(4-((2R)-4-(4-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)ethylcarbamothioyl)piperazin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonyl)benzamide(XZ-14516)

A mixture of compound 39 (12 mg), 1,1′-thiocarbonyldiimidazole (6 mg)and TEA (4.2 μL) in 2 mL DCM was stirred at room temperature for 1 hour.Then compound 35 (6.5 mg) and DIPEA (0.05 mL) were added into the abovesolution. The mixture was stirred overnight and quenched by the additionof NH₄Cl (aq). Subsequently, it was with DCM and the organic phase waswashed with water x1, brine x1, dried over Na₂SO₄, filtered andevaporated to dryness. The crude product was purified by columnchromatography using DCM and methanol as eluents to afford 6.4 mgcompound XZ14516. Yield 68%. ¹H NMR (400 MHz, CDCl₃) δ 8.90 (br s, 1H),8.37 (d, J=2.1 Hz, 1H), 8.15-8.00 (m, 1H), 7.65 (d, J=7.6 Hz, 2H),7.51-7.42 (m, 1H), 7.38-7.27 (m, 5H), 7.12-7.05 (m, 2H), 6.99 (d, J=8.4Hz, 2H), 6.87 (d, J=8.5 Hz, 1H), 6.75 (dd, J=9.0, 3.8 Hz, 2H), 6.62 (dd,J=12.9, 9.5 Hz, 1H), 6.51 (t, J=5.2 Hz, 1H), 6.22 (br s, 1H), 4.95-4.80(m, 1H), 3.90-3.61 (m, 15H), 3.48-3.41 (m, 2H), 3.30-3.20 (m, 4H),3.13-2.96 (m, 2H), 2.88-2.71 (m, 5H), 2.47-2.22 (m, 12H), 2.13-2.00 (m,4H), 1.75-1.70 (m, 1H), 1.46 (t, J=6.4 Hz, 2H), 0.99 (s, 6H) ppm.

Example 10: Synthesis of XZ-14515, XZ-14510, and XZ-14540

General Procedure for the Preparation of 41a-c

A mixture of compound 7 (1.0 equiv.), corresponding amine 40a-c (1.0equiv.), and DIPEA (2.0 equiv.) in DMF were stirred at 90° C. overnight.The mixture was poured into water and extracted with EtOAc. The organicphase was washed with water x1, brine x1, dried over Na₂SO₄, filteredand evaporated to dryness. The crude product was purified by columnchromatography using EtOAc and hexanes as eluents.

General Procedure for the Preparation of 42a-c

To a mixture of compound 41a, 41b, or 41c in DCM was added TFA. Themixture was stirred at room temperature overnight and the solvent wasremoved under reduced pressure. The crude product was washed with Et₂Oto give the corresponding acid 42a, 42b, and 42c, respectively.

Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)acetyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(XZ-14515)

A mixture of compound 35 (10 mg), 42a (5.5 mg), HATU (4 mg) and DIPEA(20 mg) in 3 mL DCM was stirred at room temperature for 1 hour. NH₄Cl(aq.) was then added to the mixture and the resulted mixture wasextracted with DCM. The organic phase was washed with water x1, brinex1, dried over Na₂SO₄, filtered and evaporated to dryness. The crudeproduct was purified by column chromatography using DCM and methanol aseluents to afford 6.6 mg pure XZ-14515. Yield 47%. ¹H NMR (400 MHz,CDCl₃ and CD₃OD) δ 9.11 (br s, 1H), 8.36 (s, 1H), 8.08 (d, J=9.1 Hz,1H), 7.66 (d, J=8.2 Hz, 2H), 7.46 (t, J=7.8 Hz, 1H), 7.39-7.27 (m, 6H),7.14-7.02 (m, 2H), 6.98 (d, J=8.3 Hz, 2H), 6.88 (d, J=8.5 Hz, 1H), 6.75(d, J=8.6 Hz, 2H), 6.58 (d, J=5.8 Hz, 1H), 6.52-6.43 (br s, 1H),4.91-4.81 (m, 1H), 4.26-4.15 (m, 2H), 3.94-3.81 (m, 1H), 3.70-3.33 (m,12H), 3.31-3.22 (m, 4H), 3.12-2.93 (m, 2H), 2.89-2.55 (m, 5H), 2.49-2.00(m, 16H), 1.74-1.70 (m, 1H), 1.51-1.45 (m, 2H), 0.98 (s, 6H) ppm.

Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)acetyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(XZ-14510)

A mixture of compound 35 (10 mg), 42b (5 mg), HATU (4 mg) and DIPEA (20mg) in 3 mL DCM was stirred at room temperature for 1 hour. NH₄Cl (aq)was then added to the mixture and the resulted mixture was extractedwith DCM. The organic phase was washed with water x1, brine x1, driedover Na₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents toafford 6.2 mg pure XZ-14510. Yield 44%. ¹H NMR (400 MHz, CDCl₃ andCD₃OD) δ 8.33-8.28 (m, 1H), 8.07 (d, J=9.0 Hz, 1H), 7.78 (d, J=8.8 Hz,2H), 7.54-7.45 (m, 1H), 7.41-7.37 (m, 1H), 7.33-7.20 (m, 5H), 7.09 (d,J=7.1 Hz, 1H), 7.06-6.98 (m, 3H), 6.94 (d, J=8.6 Hz, 1H), 6.79 (d, J=8.9Hz, 2H), 6.61 (d, J=9.4 Hz, 1H), 5.00-4.86 (m, 1H), 4.21 (s, 2H),3.93-3.84 (m, 1H), 3.70-3.39 (m, 16H), 3.32-3.25 (m, 4H), 3.12-3.00 (m,2H), 2.93-2.71 (m, 5H), 2.46-2.24 (m, 12H), 2.09-2.00 (m, 4H), 1.75-1.63(m, 1H), 1.47 (t, J=6.3 Hz, 2H), 0.99 (s, 6H) ppm.

Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(XZ-14540)

A mixture of compound 35 (12 mg), 42c (8.2 mg), HATU (5 mg) and DIPEA(30 mg) in 3 mL DCM was stirred at room temperature for 2 hours. NH₄Cl(aq.) was then added to the mixture and the resulted mixture wasextracted with DCM. The organic phase was washed with water x1, brinex1, dried over Na₂SO₄, filtered and evaporated to dryness. The crudeproduct was purified by column chromatography using DCM and methanol aseluents to afford 13.6 mg pure XZ14540. Yield 76%. ¹H NMR (400 MHz,CDCl₃ and CD₃OD) δ 8.81 (br s, 1H), 8.33 (s, 1H), 8.13-7.99 (m, 1H),7.79-7.60 (m, 2H), 7.45 (t, J=7.8 Hz, 1H), 7.38-7.26 (m, 5H), 7.24-7.17(m, 1H), 7.12-6.94 (m, 4H), 6.88 (d, J=8.6 Hz, 1H), 6.73 (d, J=8.8 Hz,2H), 6.61-6.50 (m, 1H), 6.46 (br s, 1H), 4.95-4.84 (m, 1H), 4.16 (s,2H), 3.88-3.80 (m, 1H), 3.72-3.39 (m, 20H), 3.32-3.25 (m, 4H), 3.12-3.01(m, 2H), 2.93-2.71 (m, 5H), 2.46-2.24 (m, 12H), 2.09-2.00 (m, 4H),1.75-1.63 (m, 1H), 1.47 (t, J=6.3 Hz, 2H), 0.96 (s, 6H) ppm.

Example 11: Synthesis of XZ-15416, XZ-15405, and XZ-15418

General Procedure for the Preparation of 44a-c

A mixture of compound 7 (1.0 equiv.), corresponding amine 43a-c (1.0equiv.) and DIPEA (2.0 equiv.) in DMF were stirred at 90° C. overnight.The mixture was poured into water and extracted with EtOAc. The organicphase was washed with water x1, brine x1, dried over Na₂SO₄, filteredand evaporated to dryness. The crude product was purified by columnchromatography using DCM and methanol as eluents.

Preparation of2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-hydroxyethoxy)ethyl)amino)isoindoline-1,3-dione (44a)

¹H NMR (400 MHz, CDCl₃) δ 8.25 (br s, 1H), 7.58-7.46 (m, 1H), 7.11 (d,J=7.1 Hz, 1H), 6.91 (d, J=8.5 Hz, 1H), 6.57 (t, J=5.4 Hz, 1H), 4.92 (dd,J=12.2, 5.3 Hz, 1H), 3.81-3.71 (m, 4H), 3.66-3.61 (m, 2H), 3.48 (dd,J=10.7, 5.3 Hz, 2H), 2.92-2.67 (m, 3H), 2.32 (br s, 1H), 2.18-2.07 (m,1H) ppm.

Preparation of2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-(2-hydroxyethoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione (44b)

¹H NMR (400 MHz, CDCl₃) δ 8.19 (br s, 1H), 7.55-7.44 (m, 1H), 7.10 (d,J=7.1 Hz, 1H), 6.91 (d, J=8.5 Hz, 1H), 6.57 (t, J=5.2 Hz, 1H), 4.91 (dd,J=12.0, 5.4 Hz, 1H), 3.85-3.65 (m, 8H), 3.64-3.59 (m, 2H), 3.51-3.43 (m,2H), 2.92-2.68 (m, 3H), 2.57 (br s, 1H), 2.18-2.07 (m, 1H) ppm.

Preparation of2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione (44c)

¹H NMR (400 MHz, CDCl₃) δ 8.23 (br s, 1H), 7.58-7.40 (m, 1H), 7.10 (d,J=7.1 Hz, 1H), 6.92 (d, J=8.6 Hz, 1H), 6.52 (t, J=5.5 Hz, 1H), 4.92 (dd,J=12.0, 5.4 Hz, 1H), 3.77-3.65 (m, 12H), 3.63-3.58 (m, 2H), 3.52-3.44(m, 2H), 3.00-2.59 (m, 4H), 2.24-2.04 (m, 1H) ppm.

General Procedure for the Preparation of 45a-c

To a mixture of 44a, 44b, or 44c (1.0 equiv.), TEA (4.0 equiv.) in DCMwas added MsCl (1.2 equiv.). The mixture was stirred at room temperaturefor 3 hours. Then the mixture was poured into water and extracted withEtOAc. The organic phase was washed with water x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents.

Preparation of2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethylmethanesulfonate (45a)

¹H NMR (400 MHz, CDCl₃) δ 8.10 (br s, 1H), 7.63-7.44 (m, 1H), 7.12 (d,J=7.1 Hz, 1H), 6.93 (d, J=8.5 Hz, 1H), 6.49 (t, J=5.5 Hz, 1H), 4.91 (dd,J=12.1, 5.3 Hz, 1H), 4.48-4.35 (m, 2H), 3.86-3.66 (m, 4H), 3.58-3.41 (m,2H), 3.13-2.69 (m, 6H), 2.23-2.05 (m, 1H) ppm.

Preparation of2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethylmethanesulfonate (45b)

¹H NMR (400 MHz, CDCl₃) δ 8.14 (br s, 1H), 7.65-7.45 (m, 1H), 7.12 (d,J=7.1 Hz, 1H), 6.91 (d, J=8.5 Hz, 1H), 6.51 (t, J=5.1 Hz, 1H), 4.94 (dd,J=12.0, 5.3 Hz, 1H), 4.39 (dd, J=5.3, 3.7 Hz, 2H), 4.00-3.66 (m, 8H),3.52-3.44 (m, 2H), 3.05 (s, 3H), 2.93-2.62 (m, 3H), 2.28-2.06 (m, 1H)ppm.

Preparation of2-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl methanesulfonate (45c)

¹H NMR (400 MHz, CDCl₃) δ 8.20 (br s, 1H), 7.60-7.41 (m, 1H), 7.10 (d,J=7.1 Hz, 1H), 6.92 (d, J=8.5 Hz, 1H), 6.48 (t, J=5.6 Hz, 1H), 4.92 (dd,J=11.8, 5.4 Hz, 1H), 4.36 (dd, J=5.3, 3.7 Hz, 2H), 3.82-3.60 (m, 12H),3.54-3.40 (m, 2H), 3.07 (s, 3H), 2.98-2.65 (m, 3H), 2.24-2.06 (m, 1H)ppm.

Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(XZ-15416)

A mixture of compound 35 (25 mg), 45a (12 mg), DIPEA (60 μL) and NaI(1.6 mg) in 2 mL 1,4-dioxane was heated at 90° C. overnight. Then themixture was poured into water and extracted with EtOAc. The organicphase was washed with water x1, NH₄Cl (aq) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents toafford 8.8 mg pure XZ-15416. Yield 26%. ¹H NMR (400 MHz, CDCl₃) δ 8.35(s, 1H), 8.09-7.98 (m, 1H), 7.72 (d, J=8.7 Hz, 2H), 7.48 (t, J=7.9 Hz,1H), 7.40-7.29 (m, 5H), 7.25-7.20 (m, 1H), 7.10 (d, J=7.1 Hz, 1H),7.06-6.95 (m, 3H), 6.89 (d, J=8.4 Hz, 1H), 6.73 (d, J=9.1 Hz, 2H),6.66-6.55 (m, 1H), 6.53-6.42 (m, 1H), 4.98-4.82 (m, 1H), 3.93-3.80 (m,1H), 3.76-3.40 (m, 6H), 3.32-2.64 (m, 17H), 2.43-1.97 (m, 16H),1.70-1.66 (m, 1H), 1.52-1.41 (m, 2H), 1.01-0.95 (m, 6H) ppm.

Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(XZ-15405)

A mixture of compound 35 (10 mg), 45b (6 mg), TEA (20 μL) and NaI (1.0mg) in 2 mL 1,4-dioxane was heated at 80° C. overnight. The reactionmixture was then poured into water and extracted with EtOAc. The organicphase was washed with water x1, NH₄Cl (aq) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents toafford 5.8 mg pure XZ-15405. Yield 42%. ¹H NMR (400 MHz, CDCl₃) δ 8.33(s, 1H), 8.02 (t, J=8.9 Hz, 1H), 7.76 (d, J=7.0 Hz, 2H), 7.52-7.46 (m,1H), 7.41-7.33 (m, 2H), 7.32-7.27 (m, 3H), 7.25-7.21 (m, 1H), 7.10 (dd,J=7.1, 2.3 Hz, 1H), 7.04-6.92 (m, 3H), 6.88 (d, J=8.6 Hz, 1H), 6.75 (d,J=8.4 Hz, 2H), 6.56-6.40 (m, 2H), 4.96-4.73 (m, 1H), 3.86-3.40 (m, 11H),3.33-2.51 (m, 17H), 2.50-1.79 (m, 16H), 1.74-1.60 (m, 1H), 1.48-1.37 (m,2H), 0.95 (d, J=5.4 Hz, 6H) ppm.

Preparation of4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((2R)-4-(4-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide(XZ-15418)

A mixture of compound 35 (42 mg), 45c (24 mg), DIPEA (100 μL) and NaI (3mg) in 3 mL 1,4-dioxane was heated at 90° C. overnight. The reactionmixture was then poured into water and extracted with EtOAc. The organicphase was washed with water x1, NH₄Cl (aq) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents toafford 16.4 mg pure XZ-15418. Yield 25%. ¹H NMR (400 MHz, CDCl₃) δ 8.31(s, 1H), 8.01 (d, J=8.9 Hz, 1H), 7.81 (d, J=8.5 Hz, 2H), 7.47 (t, J=7.8Hz, 1H), 7.38-7.28 (m, 5H), 7.25-7.21 (m, 1H), 7.08 (d, J=7.1 Hz, 1H),6.99 (d, J=8.3 Hz, 2H), 6.96-6.85 (m, 2H), 6.75 (d, J=8.7 Hz, 2H),6.54-6.43 (m, 2H), 4.96-4.83 (m, 1H), 3.90-3.39 (m, 15H), 3.28-2.68 (m,17H), 2.51-1.95 (m, 16H), 1.61-1.57 (m, 1H), 1.47-1.41 (m, 2H),0.97-0.93 (m, 6H) ppm.

Example 12: Synthesis of XZ-15421

Preparation of(R)—N-(4-(4-(4-(2-azidoacetyl)piperazin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenylsulfonyl)-4-(4-(2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)benzamide(46)

To a mixture of compound 35 (48 mg), 2-azidoacetic acid (8 mg), andDIPEA (13 μL) in 5 mL DCM was added HATU (21 mg). The mixture wasstirred at room temperature for 3 hours. The solvent was removed underreduced pressure and the crude product was purified via columnchromatography using DCM and methanol as eluents to afford 48 mgcompound 46. Yield 87%. ¹H NMR (400 MHz, CDCl₃) δ 8.35 (d, J=1.9 Hz,1H), 8.11 (dd, J=9.1, 1.8 Hz, 1H), 7.66 (d, J=8.8 Hz, 2H), 7.40-7.26 (m,6H), 7.08 (d, J=8.4 Hz, 1H), 6.98 (d, J=8.3 Hz, 2H), 6.76 (d, J=8.8 Hz,2H), 6.57 (d, J=9.4 Hz, 1H), 3.93-3.81 (m, 3H), 3.74-3.64 (m, 1H),3.55-3.42 (m, 1H), 3.36-3.22 (m, 6H), 3.11 (dd, J=13.8, 4.8 Hz, 1H),2.99 (dd, J=13.8, 7.5 Hz, 1H), 2.85 (s, 2H), 2.50-2.21 (m, 12H),2.20-2.08 (m, 1H), 2.03-1.96 (m, 2H), 1.75-1.61 (m, 1H), 1.46 (t, J=6.4Hz, 2H), 0.97 (s, 6H) ppm.

Preparation of4-(4-((2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(4-((2R)-4-(4-(2-(4-((2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)acetyl)piperazin-1-yl)-1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)phenyl)sulfonyl)benzamide(XZ-15421)

To a mixture of compound 46 (24.0 mg) and compound 18 (11.0 mg) in 2 mLt-BuOH-THF (1:1, v/v) under argon was added CuSO₄.5H₂O (1.15 mg) andsodium ascorbate (0.91 mg) in 0.3 mL water. The mixture was stirred at50° C. for 2 hours and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 16.4 mg XZ-15421. Yield 50%. ¹H NMR (400MHz, d₆-acetone) δ 9.91 (s, 1H), 8.34 (s, 1H), 8.10 (d, J=9.2 Hz, 1H),7.92-7.78 (m, 3H), 7.66-7.55 (m, 1H), 7.48-7.20 (m, 7H), 7.17-7.00 (m,6H), 6.90 (d, J=8.6 Hz, 2H), 6.70-6.54 (m, 1H), 5.31 (s, 2H), 5.14-5.05(m, 1H), 4.63 (s, 2H), 4.33-4.25 (m, 1H), 3.79-3.20 (m, 20H), 3.02-2.83(m, 5H), 2.58-2.11 (m, 14H), 1.87-1.82 (m, 1H), 1.48 (t, J=6.4 Hz, 2H),1.00 (s, 6H) ppm.

Example 13: Synthesis of XZ-14529

Preparation of2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazaoctadecan-18-oic acid(47)

A mixture of compound 37 (250 mg), dihydrofuran-2,5-dione (120 mg), andTEA (210 μL) in 10 mL DCM was stirred at room temperature overnight. Thereaction mixture was then poured into water and extracted with DCM. Theorganic phase was washed with 1N HCl (aq.) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness to give 320 mg compound 47.Yield 92%. ¹H NMR (400 MHz, CDCl₃) δ 3.72-3.51 (m, 8H), 3.49-3.42 (m,2H), 3.39-3.26 (m, 2H), 2.74-2.62 (m, 2H), 2.58-2.44 (m, 2H), 1.46 (s,9H) ppm.

Preparation of tert-butyl((S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-10,13-dioxo-3,6-dioxa-9,14-diazaheptadecyl)carbamate(48)

To a mixture of compound 47 (100 mg), compound 24 (190 mg), and DIPEA(167 μL) in 10 mL DCM was added 116 mg HATU. The resulted mixture wasstirred at room temperature for 2 hours before poured into water andextracted with DCM. The organic phase was washed with brine x1, driedover Na₂SO₄, filtered and evaporated to dryness to give 136 mg compound48. Yield 62%. ¹H NMR (400 MHz, CDCl₃) δ 8.67 (s, 1H), 7.53 (br s, 1H),7.40-7.29 (m, 4H), 6.97 (br s, 1H), 6.53 (br s, 1H), 5.12 (br s, 1H),4.72 (t, J=8.0 Hz, 1H), 4.62-4.44 (m, 3H), 4.33 (dd, J=15.0, 5.3 Hz,1H), 4.12-3.91 (m, 1H), 3.65-3.46 (m, 9H), 3.45-3.22 (m, 4H), 2.55-2.37(m, 8H), 2.21-2.09 (m, 1H), 1.43 (s, 9H), 0.91 (s, 9H) ppm.

Preparation ofN1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N4-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)succinamide(49)

To a mixture of compound 48 (100 mg) in 10 mL DCM was added TFA (440μL). The reaction was stirred at room temperature for 2 days. Then itwas neutralized with NaHCO₃ (aq) and extracted with DCM. The organicphase was washed with brine x1, dried over Na₂SO₄, filtered andevaporated to dryness to give the crude product which can be used in thenext step without further purification. ¹H NMR (400 MHz, CDCl₃) δ 8.67(s, 1H), 7.67-7.58 (m, 1H), 7.39-7.28 (m, 5H), 6.94 (d, J=8.2 Hz, 1H),4.70 (t, J=8.3 Hz, 1H), 4.56-4.30 (m, 4H), 4.03 (d, J=10.9 Hz, 1H),3.64-3.49 (m, 9H), 3.43-3.34 (m, 2H), 2.89 (t, J=4.9 Hz, 2H), 2.57-2.36(m, 8H), 2.20-2.13 (m, 1H), 0.95 (s, 9H) ppm.

Preparation ofN1-(2-(2-(2-(4-((R)-3-((4-(N-(4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazine-1-carboxamido)ethoxy)ethoxy)ethyl)-N4-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)succinamide(XZ-14529)

A mixture of compound 49 (26 mg) and CDI (7.7 mg) in 2 mL THF wasstirred at room temperature for 1 hour. Compound 35 (14.6 mg) and DIPEA(0.05 mL) were then added. The mixture was stirred overnight andquenched by the addition of NH₄Cl (aq.), extracted with DCM and theorganic phase was washed with water x1, brine x1, dried over Na₂SO₄,filtered and evaporated to dryness. The crude product was purified bycolumn chromatography using DCM and methanol as eluents to afford 21.1mg compound XZ-14529. Yield 85%. ¹H NMR (400 MHz, CDCl₃ and CD₃OD) δ8.70 (s, 1H), 8.32 (s, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.95-7.76 (m, 3H),7.50-7.38 (m, 2H), 7.37-7.34 (m, 4H), 7.32-7.17 (m, 5H), 7.06-6.95 (m,3H), 6.78 (d, J=8.9 Hz, 2H), 6.65 (d, J=9.4 Hz, 1H), 5.87-5.72 (m, 1H),4.66-4.47 (m, 4H), 4.45-4.33 (m, 1H), 4.01-3.22 (m, 23H), 3.16-3.03 (m,2H), 2.84 (s, 2H), 2.52-1.98 (m, 24H), 1.75-1.63 (m, 1H), 1.48 (t, J=6.3Hz, 2H), 0.99 (m, 15H) ppm.

Example 14: Synthesis of XZ-14523

Preparation of tert-butyl4-((4-(N-(2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-nitrophenylamino)methyl)piperidine-1-carboxylate(52)

A mixture of compound 50 (571 mg), 51 (415 mg), DMAP (244 mg), EDCI (250mg), and TEA (280 μL) in 20 mL DCM was stirred at room temperatureovernight. The solvent was removed under reduced pressure and theresidue was purified via column chromatography using DCM and methanol aseluents to give 758 mg pure product as yellow solid. Yield 79%. ¹H NMR(400 MHz, CDCl₃) δ 10.14 (br s, 1H), 9.72 (br s, 1H), 8.89 (d, J=2.2 Hz,1H), 8.52 (t, J=5.4 Hz, 1H), 8.21 (d, J=2.5 Hz, 1H), 8.16 (dd, J=9.2,2.1 Hz, 1H), 7.95 (d, J=9.1 Hz, 1H), 7.71 (d, J=2.5 Hz, 1H), 7.53-7.43(m, 1H), 7.22 (d, J=8.4 Hz, 2H), 6.94-6.83 (m, 3H), 6.60-6.47 (m, 2H),5.98 (d, J=2.1 Hz, 1H), 4.27-4.13 (m, 2H), 3.32-3.20 (m, 2H), 3.13-3.01(m, 4H), 2.83-2.65 (m, 4H), 2.26-2.10 (m, 6H), 1.96 (s, 2H), 1.92-1.74(m, 3H), 1.47 (s, 9H), 1.40 (t, J=6.4 Hz, 2H), 1.25-1.18 (m, 2H), 0.93(s, 6H) ppm.

Preparation of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(piperidin-4-ylmethylamino)phenylsulfonyl)benzamide(53)

To a solution of compound 52 (578 mg) in 20 mL DCM was added TFA (440μL). The reaction mixture was stirred at room temperature overnight.Solvent was removed under reduced pressure and Et₂O was added to theresidue. The precipitated solid was collected by filtration and can beused directly in the next step without further purification. ¹H NMR (400MHz, d₆-DMSO) δ 11.88-11.54 (m, 2H), 9.34 (br s, 1H), 8.66 (t, J=6.1 Hz,1H), 8.59-8.45 (m, 2H), 8.29-8.08 (m, 1H), 8.01 (d, J=2.6 Hz, 1H), 7.80(dd, J=9.2, 2.2 Hz, 1H), 7.57-7.45 (m, 3H), 7.36 (d, J=8.4 Hz, 2H), 7.12(d, J=9.4 Hz, 1H), 7.05 (d, J=8.3 Hz, 2H), 6.69 (dd, J=9.0, 2.0 Hz, 1H),6.37 (dd, J=3.3, 1.9 Hz, 1H), 6.22 (s, 1H), 3.84-3.44 (m, 4H), 3.39-3.15(m, 6H), 3.10-2.60 (m, 6H), 2.22-2.10 (m, 2H), 2.04-1.76 (m, 5H),1.49-1.26 (m, 4H), 0.91 (s, 6H) ppm.

Preparation of2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((4-(((1-(4-azidobutanoyl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzamide(54)

To a solution of compound 53 (60 mg), 4-azidobutanoic acid (7 mg), andDIPEA (42 μL) in 5 mL DCM was added HATU (20 mg). The resulted mixturewas stirred at room temperature for 2 hours. Solvent was removed underreduced pressure and the crude product was purified via columnchromatography using DCM and methanol as eluents to afford 46 mgcompound 54. Yield 94%. ¹H NMR (400 MHz, CDCl₃) δ 10.14 (br s, 1H), 9.46(s, 1H), 8.90 (d, J=2.2 Hz, 1H), 8.52 (t, J=5.4 Hz, 1H), 8.27-8.10 (m,2H), 7.95 (d, J=9.1 Hz, 1H), 7.71 (d, J=2.5 Hz, 1H), 7.51-7.44 (m, 1H),7.22 (d, J=8.4 Hz, 2H), 7.01-6.76 (m, 3H), 6.62-6.43 (m, 2H), 5.98 (d,J=2.1 Hz, 1H), 4.72 (d, J=13.5 Hz, 1H), 3.94 (d, J=13.8 Hz, 1H), 3.40(t, J=6.4 Hz, 2H), 3.34-3.20 (m, 2H), 3.13-2.98 (m, 5H), 2.74 (s, 2H),2.58 (t, J=11.7 Hz, 1H), 2.44 (t, J=7.2 Hz, 2H), 2.29-2.09 (m, 6H),2.04-1.82 (m, 7H), 1.40 (t, J=6.4 Hz, 2H), 1.28-1.18 (m, 2H), 0.93 (s,6H) ppm.

Preparation of2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-(4-(4-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)benzamide(XZ-14523)

To a mixture of compound 54 (20.0 mg), compound 9 (10.0 mg) in 2 mLt-BuOH-THF (1:1, v/v) under argon was added CuSO₄.5H₂O (1.0 mg) andsodium ascorbate (0.8 mg) in 0.3 mL water. The mixture was stirred at55° C. for 3 hours and extracted with DCM. The organic phase was washedwith brine x1, dried over Na₂SO₄, filtered and evaporated to dryness.The crude product was purified via column chromatography using DCM andmethanol as eluents to afford 23.2 mg XZ-15423. Yield 82%. ¹H NMR (400MHz, CDCl₃) δ 10.07 (br s, 1H), 9.88 (br s, 1H), 9.61 (br s, 1H), 8.89(d, J=2.2 Hz, 1H), 8.64-8.46 (m, 1H), 8.19 (d, J=2.5 Hz, 1H), 8.07 (d,J=9.2 Hz, 1H), 7.93 (d, J=9.1 Hz, 1H), 7.69 (d, J=2.5 Hz, 1H), 7.62 (s,1H), 7.52-7.42 (m, 2H), 7.23 (d, J=8.3 Hz, 2H), 7.08 (d, J=7.1 Hz, 1H),6.96-6.83 (m, 4H), 6.61-6.39 (m, 3H), 5.98 (d, J=2.0 Hz, 1H), 4.98-4.90(m, 1H), 4.76-4.63 (m, 3H), 4.53-4.37 (m, 2H), 3.88-3.62 (m, 11H),3.50-3.41 (m, 2H), 3.33-3.22 (m, 2H), 3.10-3.02 (m, 4H), 3.02-2.69 (m,6H), 2.55 (t, J=11.8 Hz, 1H), 2.38-2.11 (m, 11H), 2.00-1.80 (m, 5H),1.41 (t, J=7.3 Hz, 2H), 1.27-1.21 (m, 2H), 0.93 (s, 6H) ppm.

Example 15: Synthesis of XZ-14522 Preparation of2-((1H-pyrrolo-[2,3-b]pyridin-5-yl)oxy)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)acetyl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)benzamide(XZ-14522)

A solution of compound 53 (12 mg), compound 42b (5 mg), HATU (4 mg), andDIPEA (20 mg) in 3 mL DCM was stirred at room temperature for 2 hours.NH₄Cl (aq) was then added and extracted with DCM. The organic phase waswashed with water x1, brine x1, dried over Na₂SO₄, filtered andevaporated to dryness. The crude product was purified by columnchromatography using DCM and methanol as eluents to afford 10.8 mg pureXZ-14522. Yield 82%. ¹H NMR (400 MHz, CDCl₃) δ 10.24 (d, J=6.9 Hz, 1H),10.10 (br s, 1H), 9.82 (br s, 1H), 8.87 (s, 1H), 8.49 (t, J=5.3 Hz, 1H),8.16 (d, J=2.1 Hz, 1H), 8.07 (d, J=9.0 Hz, 1H), 7.92 (d, J=9.1 Hz, 1H),7.75-7.62 (m, 1H), 7.54-7.41 (m, 2H), 7.23 (d, J=8.3 Hz, 2H), 7.08 (d,J=7.1 Hz, 1H), 6.99-6.78 (m, 4H), 6.60-6.43 (m, 3H), 6.04-5.81 (m, 1H),5.01-4.85 (m, 1H), 4.63 (d, J=12.5 Hz, 1H), 4.39-4.00 (m, 3H), 3.75-3.64(m, 10H), 3.52-3.40 (m, 2H), 3.31-2.67 (m, 12H), 2.59 (t, J=12.4 Hz,1H), 2.27-1.81 (m, 12H), 1.46-1.38 (m, 2H), 1.26-1.24 (m, 2H), 0.93 (s,6H) ppm.

Example 16: Synthesis of XZ-14528 Preparation ofN1-(2-(2-(2-(4-(((4-(N-(2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-nitrophenyl)amino)methyl)piperidine-1-carboxamido)ethoxy)ethoxy)ethyl)-N4-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)succinamide(XZ-14528)

A mixture of compound 49 (26 mg) and CDI (7.7 mg) in 2 mL THF wasstirred at room temperature for 1 hour. Compound 53 (18.1 mg) and DIPEA(0.05 mL) were then added. The mixture was stirred overnight andquenched by the addition of NH₄Cl (aq.), extracted with DCM and theorganic phase was washed with water x1, brine x1, dried over Na₂SO₄,filtered and evaporated to dryness. The crude product was purified bycolumn chromatography using DCM and methanol as eluents to afford 16.8mg compound XZ-14528. Yield 72%. ¹H NMR (400 MHz, CDCl₃) δ 9.49 (s, 1H),8.85 (d, J=2.2 Hz, 1H), 8.67 (s, 1H), 8.57-8.43 (m, 1H), 8.15 (d, J=2.5Hz, 1H), 7.99 (d, J=9.2 Hz, 1H), 7.92 (d, J=9.1 Hz, 1H), 7.62 (d, J=2.4Hz, 1H), 7.46-7.40 (m, 2H), 7.38-7.32 (m, 4H), 7.23 (d, J=8.3 Hz, 2H),6.97-6.88 (m, 3H), 6.80 (d, J=9.4 Hz, 1H), 6.62-6.47 (m, 3H), 6.02 (d,J=1.7 Hz, 1H), 5.24-5.17 (m, 1H), 4.73 (t, J=8.0 Hz, 1H), 4.63-4.47 (m,3H), 4.35 (dd, J=15.0, 5.2 Hz, 1H), 4.10-3.89 (m, 4H), 3.64-3.47 (m,12H), 3.46-3.37 (m, 4H), 3.24 (t, J=6.1 Hz, 2H), 3.15-3.01 (m, 4H),2.87-2.71 (m, 4H), 2.60-2.38 (m, 8H), 2.20-1.76 (m, 8H), 1.42-1.34 (m,2H), 1.28-1.24 (m, 2H), 0.98-0.92 (m, 15H) ppm.

Example 17: Synthesis of XZ-15434 Preparation of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(4-((1-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethyl)piperidin-4-yl)methylamino)-3-nitrophenylsulfonyl)benzamide(XZ-15434)

A mixture of compound 53 (37 mg), compound 45a (14 mg), DIPEA (100 μL),and NaI (3 mg) in 2 mL 1,4-dioxane was heated at 90° C. overnight. Themixture was then poured into water and extracted with EtOAc. The organicphase was washed with water x1, NH₄Cl (aq.) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents toafford 10.7 mg compound XZ-15434. Yield 31%. ¹H NMR (400 MHz, CDCl₃ andCD₃OD) δ 8.77 (s, 1H), 8.43 (s, 1H), 8.04 (s, 1H), 7.94-7.83 (m, 2H),7.59 (s, 1H), 7.50 (dd, J=8.5, 7.2 Hz, 1H), 7.42 (d, J=3.4 Hz, 1H),7.26-7.17 (m, 2H), 7.09 (d, J=7.0 Hz, 1H), 6.96-6.87 (m, 3H), 6.75 (s,1H), 6.55 (dd, J=9.1, 2.1 Hz, 1H), 6.45 (d, J=2.8 Hz, 1H), 6.03 (s, 1H),5.00-4.86 (m, 1H), 3.96-3.88 (m, 2H), 3.75-3.68 (m, 2H), 3.57-3.44 (m,4H), 3.30-2.58 (m, 15H), 2.24-1.83 (m, 12H), 1.45-1.31 (m, 4H), 0.92 (s,6H) ppm.

Example 18: Synthesis of XZ-15438 Preparation of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(4-((1-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)ethyl)piperidin-4-yl)methylamino)-3-nitrophenylsulfonyl)benzamide(XZ-15438)

A mixture of compound 53 (37 mg), compound 45b (15 mg), DIPEA (100 μL),and NaI (3 mg) in 2 mL 1,4-dioxane was heated at 90° C. overnight. Themixture was then poured into water and extracted with EtOAc. The organicphase was washed with water x1, NH₄Cl (aq) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents toafford 11.7 mg compound XZ-15438. Yield 31%. ¹H NMR (400 MHz, CDCl₃ andCD₃OD) δ 10.61 (br s, 1H), 10.19 (br s, 1H), 8.81 (s, 1H), 8.39 (s, 1H),8.04 (s, 1H), 7.87 (d, J=8.9 Hz, 1H), 7.74 (s, 1H), 7.62 (s, 1H), 7.48(d, J=8.0 Hz, 2H), 7.23 (d, J=8.2 Hz, 2H), 7.06 (d, J=7.0 Hz, 1H),6.97-6.82 (m, 3H), 6.70-6.43 (m, 4H), 6.01 (s, 1H), 5.15-4.90 (m, 1H),4.17-3.83 (m, 2H), 3.78-3.58 (m, 8H), 3.48-3.40 (m, 2H), 3.21-2.73 (m,15H), 2.25-1.94 (m, 12H), 1.45-1.33 (m, 4H), 0.93 (s, 6H) ppm.

Example 19: Synthesis of XZ-15436 Preparation of2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(4-((1-(2-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)ethoxy)ethyl)piperidin-4-yl)methylamino)-3-nitrophenylsulfonyl)benzamide(XZ-15436)

A mixture of compound 53 (37 mg), compound 45c (16 mg), DIPEA (100 μL),and NaI (3 mg) in 2 mL 1,4-dioxane was heated at 90° C. overnight. Themixture was then poured into water and extracted with EtOAc. The organicphase was washed with water x1, NH₄Cl (aq) x1, brine x1, dried overNa₂SO₄, filtered and evaporated to dryness. The crude product waspurified by column chromatography using DCM and methanol as eluents toafford 15.0 mg compound XZ-15436. Yield 39%. ¹H NMR (400 MHz, CDCl₃ andCD₃OD) δ 10.59 (br s, 1H), 10.06 (br s, 1H), 8.83 (d, J=2.2 Hz, 1H),8.43 (t, J=5.4 Hz, 1H), 8.11 (d, J=2.3 Hz, 1H), 7.90 (d, J=9.1 Hz, 2H),7.63 (d, J=2.4 Hz, 1H), 7.54-7.42 (m, 2H), 7.22 (d, J=8.4 Hz, 2H), 7.07(d, J=7.1 Hz, 1H), 6.98-6.84 (m, 3H), 6.73 (d, J=9.2 Hz, 1H), 6.61-6.45(m, 3H), 6.01 (d, J=2.0 Hz, 1H), 5.01-4.93 (m, 1H), 3.94-3.79 (m, 2H),3.76-3.61 (m, 10H), 3.54-3.40 (m, 4H), 3.27-3.17 (m, 2H), 3.16-2.43 (m,13H), 2.28-2.09 (m, 7H), 1.98-1.82 (m, 5H), 1.41 (t, J=6.4 Hz, 2H),1.26-1.23 (m, 2H), 0.93 (s, 6H) ppm.

Example 20: Synthesis of XZ-14548 Preparation of1-((R)-3-((4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperidin-4-yl(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate(XZ-14548)

A mixture of BM1197 (11.5 mg) and CDI (10 mg) in 1.5 mL dichloroethanewas heated at 60° C. overnight. A solution of compound 39 (5.5 mg) andTEA (0.3 mL) in 0.5 mL DMSO was then added and the resulted mixture washeated to 70° C. overnight. After cooled to room temperature, themixture was poured into water and extracted with EtOAc. The organicphase was washed with water x1, brine x1, dried over Na₂SO₄, filteredand evaporated to dryness. The crude product was purified by columnchromatography using DCM and methanol as eluents to afford 3.6 mgcompound XZ-14548. Yield 22%. ¹H NMR (400 MHz, CDCl₃) δ 8.79 (br s, 1H),7.99 (s, 1H), 7.65-7.56 (m, 1H), 7.54-7.46 (m, 1H), 7.37-7.27 (m, 4H),7.26-7.20 (m, 2H), 7.14-7.05 (m, 3H), 7.03-6.94 (m, 3H), 6.91 (d, J=8.4Hz, 1H), 6.81 (d, J=8.8 Hz, 2H), 6.70 (s, 1H), 6.66-6.50 (m, 2H), 6.43(d, J=11.9 Hz, 1H), 6.30 (d, J=8.7 Hz, 1H), 5.34-5.26 (m, 1H), 4.98-4.88(m, 1H), 4.73-4.55 (m, 1H), 4.48-4.34 (m, 1H), 3.96-3.81 (m, 1H),3.75-3.37 (m, 12H), 3.22-3.03 (m, 10H), 2.96-2.61 (m, 10H), 2.57-2.43(m, 1H), 2.40-1.63 (m, 11H), 1.45 (d, J=7.1 Hz, 6H) ppm.

Example 21: Evaluation of Compounds of Formula (I) and Formula (II) fortheir Ability to Selectively Kill Senescent Cells

Normal WI38 (NSC) and IR-induced senescent WI38 cells (IR-SC) wereincubated with vehicle or increasing concentrations of compounds ofFormula (I) or Formula (II) for 72 hours. The cells were digested with0.25% trypsin and 1 mM EDTA, and harvested in PBS containing 2% FBS.After incubation with propidium iodide (PI, 100 ng/ml) in PBS at roomtemperature for 1 minute, cells were centrifuged at 1,200 rpm for 6minutes to remove PI and then resuspended in PBS containing 2% FBS foranalysis using a flow cytometer. Viable cells (PI− cells) were analyzedby flow cytometry at a constant flow rate to count the number of cellsand calculated as a percentage of control cells treated with vehicle.Table 1 depicts the EC50 values of the compounds of Formula (I) andcompounds of Formula (II) against normal WI38 and IR-induced senescentWI38.

Both compound 11 (XZ-13861) (FIG. 2A) and XZ-12906 (FIG. 2B) selectivelyinhibit IR-SC WI38 cells but not normal WI 38 cells in a dose dependentmanner.

Example 22: Evaluation of Compounds of Formula (I) and Formula (II) fortheir Ability to Kill Cancer Cells

RS4; 11 and NCI-H146 cancer cells were incubated with vehicle orincreasing concentrations of compounds of Formula (I) and Formula (II).At 72 hours post-treatment cell viability was measured by MTS and EC50was calculated as a percentage of control cells treated with vehicle.Table 1 depicts the EC50 values of compounds of Formula (I) and Formula(II) against RS4; 11 and NCI-H146.

TABLE 1 EC50 values of compounds of Formula (I) and Formula (II) againstRS4; 11 and NCI-H146. WI38 EC50 (μM) Ratio EC50 (nM) Compound NSC IR-SCNSC/SC RS4 NCI-H146 ABT-263 12.6 0.61 20.6 16.00 27.47 XZ-14439 >200.87 >20.88 84.43 69.55 PZ-15227 >10 0.108 >92 113.62 68.86 XZ-15421 >10<0.1562 >64.02 72.92 XZ-14510 >10 0.177 >56 219.89 111.98 XZ-14509 >100.226 >44.22 90.00 64.95 XZ-14515 >10 0.158 >63.27 173.16 79.17XZ-14516 >10 0.092 >109 1088.63 517.16 XZ-14540 >10 1.13 8.86 82.4376.25 XZ-14437 >20 4.55 >4.4 460 >250 XZ-14529 >10 >10 — 822.56 >2000XZ-15416 >10 <0.1562 >64.02 30.71 >10 XZ-15405 >10 0.152 >65.97 24.78XZ-15418 >10 0.172 >58.06 12.33 ABT-199 >10 >10 — XZ-14522 >10 >10 —46.98 1103.7 XZ-14523 >10 >10 — 160.86 1255.91 XZ-14528 >10 >10 —396.62 >2000 XZ-13906 100 2.4 41.67 >2000 >2000 XZ-14455 5.0 1.473.40 >2000 >2000 XZ-14424 1.6 1.4 1.2 XZ-13861 32.8 3.12 10.51

Example 23: Protein Degradation Assays in Senescent Cells

IR-SC WI38 cells were incubated with vehicle or increasingconcentrations of XZ-14439 for 18 hours at increasing contractions (FIG.3A) and at a fixed concentration of XZ-14439 for increasing times (FIG.3B). The cells were digested with 0.25% trypsin and 1 mM EDTA, andharvested in RIPA lysis buffer with 1 Phosphatase Inhibitor Cocktail 3and 1% Protease Inhibitor Cocktail. An equal amount of protein (15-30μg/lane) from each cell extract was resolved on a 12% SDS-PAGE gel.Proteins were blotted onto a NOVEX PVDF membrane by electrophoresis. Themembranes were blocked with TBS-T blocking buffer (5% nonfat milk in 25mM Tris-HCL, pH 7.4; 3 mM KCl; 1n40 mM NaCl; and 0.05% Tween) and probedwith primary antibodies (at a predetermined optimal concentration)overnight at 4° C. or for 1 hour at room temperature. After extensivewashing with TBS-T, the membranes were incubated with an appropriateperoxidase-conjugated secondary antibody for 1 hour at room temperature.After three washes with TBS-T, the proteins of interest were detectedwith ECL Western Blotting Detection Reagents and recorded withautoradiography (Pierce Biotech, Rockford, Ill., USA). The primaryantibody Bcl-xl (#2762), Bcl-2 antibody (#2872S) and β-actin (13E5,#4970) were purchased from Cell Signaling.

XZ-14439 depletes Bcl-xL in IR-SC WI38 cells in both a dose dependent(FIG. 3A) and time dependent (FIG. 3B) manner.

Example 24: Protein Degradation Assays in Cancer Cells

IR-SC WI38 (FIG. 4A) and RS4; 11 (FIG. 4B) cells were incubated withvehicle or increasing concentrations of compounds of Formula (I) orFormula (II) for 6 or 16 hours, at 1 μM (FIG. 4A) and 100 nm (FIG. 4B),respectively. The cells were harvested in RIPA lysis buffer with 1%Phosphatase Inhibitor Cocktail 3 and 1 Protease Inhibitor Cocktail. Anequal amount of protein (15-30 μg/lane) from each cell extract wasresolved on a 12% SDS-PAGE gel. Proteins were blotted onto a NOVEX PVDFmembrane by electrophoresis. The membranes were blocked with TBS-Tblocking buffer (5% nonfat milk in 25 mM Tris-HCL, pH 7.4; 3 mM KCl; 140mM NaCl; and 0.05% Tween) and probed with primary antibodies (at apredetermined optimal concentration) overnight at 4° C. or for 1 hour atroom temperature. After extensive washing with TBS-T, the membranes wereincubated with an appropriate peroxidase-conjugated secondary antibody(Jackson ImmunoResearch Europe, Suffolk, UK) for 1 hour at roomtemperature. After three washes with TBS-T, the proteins of interestwere detected with ECL Western Blotting Detection Reagents and recordedwith autoradiography. The primary antibody Bcl-xl (#2762), Bcl-2antibody (#2872S), Bcl-w (#2724S), Mcl-1 (#5453s) and β-actin (13E5,#4970) were purchased from Cell Signaling.

XZ-15416, XZ-15405, XZ-15418, XZ-15421, and PZ-15227 deplete Bcl-xL inIR-SC WI38 and RS4; 11 cells at 1 μM (FIG. 4A) and 200 nM (FIG. 4B).

REFERENCES

-   Aguilar, A., et al., (2013) A potent and highly efficacious    Bcl-2/Bcl-xL inhibitor, J Med Chem 56: 3048-3067.-   Bai. L., et al. (2014) BM-1197: a novel and specific Bcl-2/Bcl-xL    inhibitor inducing complete and long-lasting tumor regression in    vivo, PLOS One 9:e99404.-   Baker, D. J., et al., (2011) Clearance of p16Ink4a-positive    senescent cells delays ageing-associated disorders, Nature    479:232-236.-   Baker, D. J., et al., (2016) Naturally occurring p16(Ink4a)-positive    cells shorten healthy lifespan, Nature 530:184-189.-   Bajwa, N., et al., (2012) Inhibitors of the anti-apoptotic Bcl-2    proteins: a patent review, Expert Opin. Ther. Patents 22:37-55.-   Bruncko, M., et al., (2007) Studies leading to potent, dual    inhibitors of Bcl-2 and Bcl-xL, J Med Chem 50:641-662.-   Bruncko, M., et al., (2015) Structure-guided design of a series of    MCL-1 inhibitors with high affinity and selectivity, J Med Chem    58:2180-2194.-   Campisi, J., (2005) Senescent cells, tumor suppression, and    organismal aging: good citizens, bad neighbors, Cell 120:513-522.-   Campisi, J., (2011) Cellular senescence: putting the paradoxes in    perspective, Curr. Opin. Genet. Dev. 21:107-112.-   Chang, J., et al., (2016) Clearance of senescent cells by ABT263    rejuvenates aged hematopoietic stem cells in mice, Nat. Med.    22:78-83.-   Chen, J., et al., (2012) Structure-based discovery of BM-957 as a    potent small-molecule inhibitor of Bcl-2 and Bcl-xL capable of    achieving complete tumor regression, J Med Chem 55:8502-8514.-   Delbridge, A. R., et al., (2016) Thirty years of BCL-2: translating    cell death discoveries into novel cancer therapies, Nat. Rev. Cancer    16:99-109.-   Jing, L., et al., (2015) Hijacking the E3 Ubiquitin Ligase Cereblon    to Efficiently Target BRD4, Chem. Biol. 22:755-763, 2015-   Rodier, F. and Campisi, J., (2011) Four faces of cellular    senescence, J. Cell Biol. 192:547-556.-   Park. C. M., et al., (2008) Discovery of an Orally Bioavailable    Small Molecule Inhibitor of Prosurvival B-Cell Lymphoma 2 Proteins,    J Med Chem 51:6902-6915.-   Pelz, N. F., et al., (2016) Discovery of 2-Indole-acylsulfonamide    Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based    Methods, J Med Chem 59:2054-2066.-   Sleebs, B. E., et al., Quinazoline Sulfonamides as Dual Binders of    the Proteins B-Cell Lymphoma 2 and B-Cell Lymphoma Extra Long with    Potent Proapoptotic Cell-Based Activity (2011) J Med Chem    54:1914-1916.-   Sleebs, B. E., et al., (2013) Discovery of Potent and Selective    Benzothiazole Hydrazone Inhibitors of Bcl-X_(L) , J Med Chem    56:5514-5540.-   Tanaka, Y., et al., (2013) Discovery of potent Mcl-1/Bcl-xL dual    inhibitors by using a hybridization strategy based on structural    analysis of target proteins, J Med Chem 56:9635-9645.-   Tao, Z. F., et al., (2014) Discovery of a Potent and Selective    BCL-XL Inhibitor with in Vivo Activity, ACS Med Chem Lett    5:1088-1093.-   Zhou, H., et al., (2012) Structure-based design of potent    Bcl-2/Bcl-xL inhibitors with strong in vivo antitumor activity, J    Med Chem 55:6149-6161.-   Lessene, G., et al., (2008) BCL-2 family antagonists for cancer    therapy, Nat. Rev. Drug Discov. 7:989-1000.-   Vogler, M., et al., (2009) Bcl-2 inhibitors: small molecules with a    big impact on cancer therapy, Cell Death Differ. 16:360-367-   Vogler, M., (2014) Targeting BCL2-Proteins for the Treatment of    Solid Tumours, Adv. Med. 1-14.-   Zhu, Y., et al., (2015) The Achilles' heel of senescent cells: from    transcriptome to senolytic drugs, Aging Cell 14:644-658.

1. A compound comprising Formula (II):

wherein R¹ is selected from the group consisting of:

R³ is absent, a bond, or a substituted or unsubstituted C₁-C₁₀ alkyl; Ais absent, a bond, a substituted or unsubstituted C₁-C₆ aryl, asubstituted or unsubstituted C₁-C₆ cycloalkyl, or a substituted orunsubstituted C₁-C₆ heterocyclic group; R⁴ is a bond or a substituted orunsubstituted C₁-C₁₀ alkyl; n is an integer from 0 to 5, R² is selectedfrom the group consisting of


2. The compound of claim 1, wherein R¹ is selected from the groupconsisting of:


3. The compound of claim 1, wherein R³ is absent, an unsubstituted C₁-C₆alkyl, or a substituted or unsubstituted C₃-C₆ ketone.
 4. The compoundof claim 3, wherein R³ is absent, a bond, an unsubstituted C₁-C₃ alkyl,or an unsubstituted C₃-C₆ ketone.
 5. The compound of claim 4, wherein R³is absent, a bond, 2-pentanone, or an unsubstituted C₂-C₃ alkyl.
 6. Thecompound of claim 1, wherein A is absent, a bond, or a substituted orunsubstituted C₁-C₆ heterocyclic group
 7. The compound of claim 6,wherein A is absent, a bond, or an unsubstituted C₅ heterocyclic group8. The compound of claim 7, wherein A is absent, a bond, or a triazole.9. The compound of claim 1, wherein n is 0 to
 3. 10. The compound ofclaim 9, wherein n is 0 to
 2. 11. The compound of claim 10, wherein n is1 to
 2. 12. The compound of claim 1, wherein R⁴ is a bond or asubstituted or unsubstituted C₁-C₁₀ alkyl.
 13. The compound of claim 12,wherein R⁴ is a bond or a substituted C₁-C₁₀ alkyl.
 14. The compound ofclaim 1, wherein R² is selected from the group consisting of:


15. The compound of claim 1, wherein the compound of Formula (II) isselected from the group consisting of:

16.-23. (canceled)
 24. A method of killing a cancer cell in a subject inneed thereof, the method comprising administering to the subject acomposition comprising a therapeutically effective amount of acomposition comprising a compound of Formula (II):

wherein R¹ is selected from the group consisting of:

R³ is absent, a bond, or a substituted or unsubstituted C₁-C₁₀ alkyl; Ais absent, a bond, a substituted or unsubstituted C₁-C₆ aryl, asubstituted or unsubstituted C₁-C₆ cycloalkyl, or a substituted orunsubstituted C₁-C₆ heterocyclic group; R⁴ is a bond or a substituted orunsubstituted C₁-C₁₀ alkyl; n is an integer from 0 to 5, R² is selectedfrom the group consisting of


25. The method of claim 24, wherein R¹ is selected from the groupconsisting of:


26. The method of claim 24, wherein R³ is absent, an unsubstituted C₁-C₆alkyl, or a substituted or unsubstituted C₃-C₆ ketone.
 27. The method ofclaim 24, wherein A is absent, a bond, or a substituted or unsubstitutedC₁-C₆ heterocyclic group.